Methods and compositions for treating complement-associated disorders

ABSTRACT

The present disclosure relates to, inter alia, compositions containing an inhibitor of human complement and use of the compositions in methods for treating or preventing complement-associated disorders. In some embodiments, the inhibitor is chronically administered to patients. In some embodiments, the inhibitor is administered to a patient in an amount and with a frequency to maintain systemic complement inhibition and prevent breakthrough. In some embodiments, the compositions contain an antibody, or antigen-binding fragment thereof, that binds to a human complement component C5 protein or a fragment of the protein such as C5a or C5b.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application No. PCT/US2009/063929, filed on Nov. 10, 2009,which claims the benefit of the filing date under 35 U.S.C. 119(e) toU.S. Provisional Patent Application Ser. No. 61/198,803, filed on Nov.10, 2008; 61/199,563, filed on Nov. 18, 2008; 61/199,562, filed on Nov.18, 2008; 61/199,569, filed on Nov. 18, 2008; 61/199,764, filed on Nov.19, 2008; 61/200,640, filed on Dec. 1, 2008; 61/200,634, filed on Dec.1, 2008; 61/200,635, filed on Dec. 1, 2008; 61/181,788, filed on May 28,2009; and 61/228,047, filed on Jul. 23, 2009, the entire content ofwhich are hereby incorporated by reference. International ApplicationNo. PCT/US2009/063929 was published under PCT Article 21(2) in English.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on May 15, 2012, is namedALXN136301Seq.txt and is 61,879 bytes in size.

TECHNICAL FIELD

The field of the invention is medicine, immunology, molecular biology,and protein chemistry.

BACKGROUND

The complement system acts in conjunction with other immunologicalsystems of the body to defend against intrusion of cellular and viralpathogens. There are at least 25 complement proteins, which are found asa complex collection of plasma proteins and membrane cofactors. Theplasma proteins make up about 10% of the globulins in vertebrate serum.Complement components achieve their immune defensive functions byinteracting in a series of intricate but precise enzymatic cleavage andmembrane binding events. The resulting complement cascade leads to theproduction of products with opsonic, immunoregulatory, and lyticfunctions. A concise summary of the biologic activities associated withcomplement activation is provided, for example, in The Merck Manual,16^(th) Edition.

The complement cascade can progress via the classical pathway (CP), thelectin pathway, or the alternative pathway (AP). The lectin pathway istypically initiated with binding of mannose-binding lectin (MBL) to highmannose substrates. The AP can be antibody independent, and can beinitiated by certain molecules on pathogen surfaces. The CP is typicallyinitiated by antibody recognition of, and binding to, an antigenic siteon a target cell. These pathways converge at the C3 convertase—the pointwhere complement component C3 is cleaved by an active protease to yieldC3a and C3b.

The AP C3 convertase is initiated by the spontaneous hydrolysis ofcomplement component C3, which is abundant in the plasma in the blood.This process, also known as “tickover,” occurs through the spontaneouscleavage of a thioester bond in C3 to form C3i or C3(H₂O). Tickover isfacilitated by the presence of surfaces that support the binding ofactivated C3 and/or have neutral or positive charge characteristics(e.g., bacterial cell surfaces). This formation of C3(H₂O) allows forthe binding of plasma protein Factor B, which in turn allows Factor D tocleave Factor B into Ba and Bb. The Bb fragment remains bound to C3 toform a complex containing C3(H₂O)Bb—the “fluid-phase” or “initiation” C3convertase. Although only produced in small amounts, the fluid-phase C3convertase can cleave multiple C3 proteins into C3a and C3b and resultsin the generation of C3b and its subsequent covalent binding to asurface (e.g., a bacterial surface). Factor B bound to the surface-boundC3b is cleaved by Factor D to thus form the surface-bound AP C3convertase complex containing C3b,Bb. (See, e.g., Müller-Eberhard (1988)Ann Rev Biochem 57:321-347.)

The AP C5 convertase—(C3b)₂,Bb—is formed upon addition of a second C3bmonomer to the AP C3 convertase. (See, e.g., Medicus et al. (1976) J ExpMed 144:1076-1093 and Fearon et al. (1975) J Exp Med 142:856-863.) Therole of the second C3b molecule is to bind C5 and present it forcleavage by Bb. (See, e.g., Isenman et al. (1980) J Immunol124:326-331.) The AP C3 and C5 convertases are stabilized by theaddition of the trimeric protein properdin as described in, e.g.,Medicus et al. (1976), supra. However, properdin binding is not requiredto form a functioning alternative pathway C3 or C5 convertase. (See,e.g., Schreiber et al. (1978) Proc Natl Acad Sci USA 75: 3948-3952 andSissons et al. (1980) Proc Natl Acad Sci USA 77: 559-562.)

The CP C3 convertase is formed upon interaction of complement componentC1, which is a complex of C1q, C1r, and C1s, with an antibody that isbound to a target antigen (e.g., a microbial antigen). The binding ofthe C1q portion of C1 to the antibody-antigen complex causes aconformational change in C1 that activates C1r. Active C1r then cleavesthe C1-associated C1s to thereby generate an active serine protease.Active C1s cleaves complement component C4 into C4b and C4a. Like C3b,the newly generated C4b fragment contains a highly reactive thiol thatreadily forms amide or ester bonds with suitable molecules on a targetsurface (e.g., a microbial cell surface). C1s also cleaves complementcomponent C2 into C2b and C2a. The complex formed by C4b and C2a is theCP C3 convertase, which is capable of processing C3 into C3a and C3b.The CP C5 convertase—C4b, C2a, C3b—is formed upon addition of a C3bmonomer to the CP C3 convertase. (See, e.g., Müller-Eberhard (1988),supra and Cooper et al. (1970) J Exp Med 132:775-793.)

In addition to its role in C3 and C5 convertases, C3b also functions asan opsonin through its interaction with complement receptors present onthe surfaces of antigen-presenting cells such as macrophages anddendritic cells. The opsonic function of C3b is generally considered tobe one of the most important anti-infective functions of the complementsystem. Patients with genetic lesions that block C3b function are proneto infection by a broad variety of pathogenic organisms, while patientswith lesions later in the complement cascade sequence, i.e., patientswith lesions that block C5 functions, are found to be more prone only toNeisseria infection, and then only somewhat more prone.

The AP and CP C5 convertases cleave C5, which is a 190 kDa beta globulinfound in normal serum at approximately 75 μg/ml (0.4 μM). C5 isglycosylated, with about 1.5-3 percent of its mass attributed tocarbohydrate. Mature C5 is a heterodimer of a 999 amino acid 115 kDaalpha chain that is disulfide linked to a 655 amino acid 75 kDa betachain. C5 is synthesized as a single chain precursor protein product ofa single copy gene (Haviland et al. (1991) J. Immunol. 146:362-368). ThecDNA sequence of the transcript of this gene predicts a secreted pro-C5precursor of 1658 amino acids along with an 18 amino acid leadersequence (see, e.g., U.S. Pat. No. 6,355,245).

The pro-C5 precursor is cleaved after amino acids 655 and 659, to yieldthe beta chain as an amino terminal fragment (amino acid residues +1 to655 of the above sequence) and the alpha chain as a carboxyl terminalfragment (amino acid residues 660 to 1658 of the above sequence), withfour amino acids (amino acid residues 656-659 of the above sequence)deleted between the two.

C5a is cleaved from the alpha chain of C5 by either alternative orclassical C5 convertase as an amino terminal fragment comprising thefirst 74 amino acids of the alpha chain (i.e., amino acid residues660-733 of the above sequence). Approximately 20 percent of the 11 kDamass of C5a is attributed to carbohydrate. The cleavage site forconvertase action is at, or immediately adjacent to, amino acid residue733 of the above sequence. A compound that would bind at, or adjacent,to this cleavage site would have the potential to block access of the C5convertase enzymes to the cleavage site and thereby act as a complementinhibitor.

C5 can also be activated by means other than C5 convertase activity.Limited trypsin digestion (see, e.g., Minta and Man (1997) J Immunol.119:1597-1602 and Wetsel and Kolb (1982) J Immunol. 128:2209-2216) andacid treatment (Yamamoto and Gewurz (1978) J Immunol. 120:2008 andDamerau et al. (1989) Molec. Immunol. 26:1133-1142) can also cleave C5and produce active C5b.

Cleavage of C5 releases C5a, a potent anaphylatoxin and chemotacticfactor, and leads to the formation of the lytic terminal complementcomplex, C5b-9. C5a and C5b-9 also have pleiotropic cell activatingproperties, by amplifying the release of downstream inflammatoryfactors, such as hydrolytic enzymes, reactive oxygen species,arachidonic acid metabolites and various cytokines.

The first step in the formation of the terminal complement complexinvolves the combination of C5b with C6, C7, and C8 to form the C5b-8complex at the surface of the target cell. Upon the binding of the C5b-8complex with several C9 molecules, the membrane attack complex (MAC,C5b-9, terminal complement complex—TCC) is formed. When sufficientnumbers of MACs insert into target cell membranes the openings theycreate (MAC pores) mediate rapid osmotic lysis of the target cells.Lower, non-lytic concentrations of MACs can produce other effects. Inparticular, membrane insertion of small numbers of the C5b-9 complexesinto endothelial cells and platelets can cause deleterious cellactivation. In some cases activation may precede cell lysis.

As mentioned above, C3a and C5a are anaphylatoxins. These activatedcomplement components can trigger mast cell degranulation, whichreleases histamine from basophils and mast cells, and other mediators ofinflammation, resulting in smooth muscle contraction, increased vascularpermeability, leukocyte activation, and other inflammatory phenomenaincluding cellular proliferation resulting in hypercellularity. C5a alsofunctions as a chemotactic peptide that serves to attractpro-inflammatory granulocytes to the site of complement activation.

C5a receptors are found on the surfaces of bronchial and alveolarepithelial cells and bronchial smooth muscle cells. C5a receptors havealso been found on eosinophils, mast cells, monocytes, neutrophils, andactivated lymphocytes.

While a properly functioning complement system provides a robust defenseagainst infecting microbes, inappropriate regulation or activation ofcomplement has been implicated in the pathogenesis of a variety ofdisorders including, e.g., rheumatoid arthritis (RA); lupus nephritis;ischemia-reperfusion injury; atypical hemolytic uremic syndrome (aHUS);dense deposit disease (DDD); macular degeneration (e.g., age-relatedmacular degeneration (AMD)); hemolysis, elevated liver enzymes, and lowplatelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP);spontaneous fetal loss; Pauci-immune vasculitis; epidermolysis bullosa;recurrent fetal loss; multiple sclerosis (MS); traumatic brain injury;and injury resulting from myocardial infarction, cardiopulmonary bypassand hemodialysis. (See, e.g., Holers et al. (2008) Immunological Reviews223:300-316.)

SUMMARY

The present disclosure relates to compositions containing an inhibitorof human complement (e.g., an inhibitor of complement component C5 suchas an anti-C5 antibody) and methods for using the compositions to treator prevent complement-associated disorders. In some embodiments, thecompositions contain an antibody, or antigen-binding fragment thereof,that binds to a human complement component C5 protein. In someembodiments, the compositions contain an antibody, or antigen-bindingfragment thereof, that binds to human C5 fragment C5a or C5b. In someembodiments, the C5 inhibitor is a small molecule or a nucleic acid suchas, e.g., a siRNA or an anti-sense RNA that binds to and promotesinactivation of C5 mRNA in a mammal.

Complement-associated disorders include any medical disorder in a human,the treatment of which would benefit directly or indirectly frominhibition of the complement system. The disorders are generallycharacterized by inappropriate regulation of the complement system suchas inappropriate: (i) activation of the complement system or (ii)duration of an activated complement system in a subject.Complement-associated disorders include, without limitation,inflammatory and autoimmune disorders. A complement-associated disordercan be, e.g., RA; antiphospholipid antibody syndrome (APS); lupusnephritis; ischemia-reperfusion injury; aHUS; typical (also referred toas diarrheal or infectious) hemolytic uremic syndrome (tHUS); DDD;neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); MS;macular degeneration (e.g., AMD); HELLP syndrome; TTP; spontaneous fetalloss; Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetalloss; and traumatic brain injury. In some embodiments, thecomplement-associated disorder is a complement-associated vasculardisorder such as a cardiovascular disorder, myocarditis, acerebrovascular disorder, a peripheral (e.g., musculoskeletal) vasculardisorder, a renovascular disorder, a mesenteric/enteric vasculardisorder, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupuserythematosus-associated vasculitis, vasculitis associated withrheumatoid arthritis, immune complex vasculitis, Takayasu's disease,dilated cardiomyopathy, diabetic angiopathy, Kawasaki's disease(arteritis), venous gas embolus (VGE), and restenosis following stentplacement, rotational atherectomy, and percutaneous transluminalcoronary angioplasty (PTCA). Additional complement-associated disordersinclude, without limitation, myasthenia gravis (MG), cold agglutinindisease (CAD), dermatomyositis, paroxysmal cold hemoglobinuria (PCH),Graves' disease, atherosclerosis, Alzheimer's disease, systemicinflammatory response sepsis, septic shock, spinal cord injury,glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis,pemphigus, autoimmune hemolytic anemia (AIHA), idiopathicthrombocytopenic purpura (ITP), Goodpasture syndrome, Degos disease, andcatastrophic APS (CAPS).

In one aspect, the disclosure features a method for treating orpreventing a complement-associated disorder in a human. The methodincludes administering to a human in need thereof a therapeuticallyeffective amount of a composition comprising an inhibitor of humancomplement (e.g., an inhibitor of human complement component C5).

In another aspect, the disclosure features a method for treating orpreventing a complement-associated disorder in a human, which methodcomprises administering to a human in need thereof a compositioncomprising a therapeutically effective amount of an inhibitor of humancomplement (e.g., an inhibitor of human complement component C5).

In some embodiments of any of the methods described herein, theinhibitor can inhibit the expression of a human complement component C5protein. The inhibitor can inhibit the protein expression of a humancomplement component C5 protein or inhibit the expression of an mRNAencoding the protein. In some embodiments of any of the methodsdescribed herein, the inhibitor can inhibit the cleavage of humancomplement component C5 into fragments C5a and C5b.

In some embodiments of any of the methods described herein, theinhibitor binds to, and inhibits, one or both of C5a and C5b. Theinhibitor can be, e.g., an antibody that binds to C5a or C5b. In someembodiments, the inhibitor is an antibody that binds to C5a, but doesnot bind to full-length C5. In some embodiments, the inhibitor is anantibody that binds to C5b, but does not bind to full-length C5. In someembodiments, the inhibitor is an antibody that binds to a human C5aprotein or a fragment thereof having an amino acid sequence thatcontains, or consists of, at least four (e.g., at least four, five, six,seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, or 17 or more)consecutive amino acids depicted in any one of SEQ ID NOs:12-25. In someembodiments, the inhibitor is an antibody that binds to human C5aprotein having the amino acid sequence depicted in SEQ ID NO:12. In someembodiments, the inhibitor is an antibody that binds to a human C5bprotein or fragment thereof having an amino acid sequence that contains,or consists of, at least four (e.g., at least four, five, six, seven,eight, nine, 10, 11, 12, 13, 14, 15, 16, or 17 or more) consecutiveamino acids depicted in any one of SEQ ID NOs:4 or 26. In someembodiments, the inhibitor is an antibody that binds to human C5bprotein having the amino acid sequence depicted in SEQ ID NO:4 or 26.

In some embodiments of any of the methods described herein, theinhibitor can be selected from the group consisting of a polypeptide, apolypeptide analog, a nucleic acid, a nucleic acid analog, and a smallmolecule. The polypeptide can be, or consist of, an antibody, orantigen-binding fragment thereof, that binds to a human complementcomponent C5 protein such as any of those described herein. In someembodiments, the antibody can bind to the alpha chain of the complementcomponent C5 protein. In some embodiments, the antibody can bind to thebeta chain of the complement component C5 protein. In some embodiments,the antibody can bind to the alpha chain of human complement componentC5, and the antibody can (i) inhibit complement activation in a humanbody fluid, (ii) inhibit the binding of purified human complementcomponent C5 to either human complement component C3b or humancomplement component C4b, and/or (iii) not bind to the human complementactivation product free C5a (or a combination of any of the foregoingproperties). The antibody can bind to the human complement component C5protein having, or consisting of, the amino acid sequence depicted inany one of SEQ ID NO s:1-11. The antibody can bind to an isolatedoligopeptide comprising an amino acid sequence corresponding to aminoacid position 8 through amino acid position 12 of SEQ ID NO:5. In someembodiments, the antibody can be a monoclonal antibody, a single-chainantibody, a humanized antibody, a fully human antibody, a polyclonalantibody, a recombinant antibody, a diabody, a chimerized or chimericantibody, a deimmunized human antibody, a fully human antibody, a singlechain antibody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab′fragment, or an F(ab′)₂ fragment. In some embodiments, the antibody canbe eculizumab or pexelizumab.

In some embodiments of any of the methods described herein, thecomposition can be intravenously administered to the human.

In some embodiments of any of the methods described herein, thecomplement-associated disorder is an alternative complementpathway-associated disorder. In some embodiments of any of the methodsdescribed herein, the complement-associated disorder is a classicalcomplement pathway-associated disorder. In some embodiments, thecomplement-associated disorder is selected from the group consisting ofrheumatoid arthritis, ischemia-reperfusion injury, atypical hemolyticuremic syndrome, thrombotic thrombocytopenic purpura, dense depositdisease, age-related macular degeneration, spontaneous fetal loss,Pauci-immune vasculitis, epidermolysis bullosa, recurrent fetal loss,multiple sclerosis, HELLP, pre-eclampsia, traumatic brain injury,Alzheimer's disease, myasthenia gravis, cold agglutinin disease,dermatomyositis, Graves' disease, Hashimoto's thyroiditis, type Idiabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathicthrombocytopenic purpura, Goodpasture syndrome, antiphospholipidsyndrome, catastrophic antiphospholipid syndrome, neuromyelitis optica(NMO), multifocal motor neuropathy (MMN), Degos disease, and any othercomplement-associated disorder described herein.

In some embodiments, any of the methods described herein can furtherinclude the step of identifying the human as having, suspected ofhaving, or at risk for developing, a complement-associated disorder. Insome embodiments, any of the methods described herein can also include,after the administering, monitoring the human for an improvement in oneor more symptoms of the complement-associated disorder.

In embodiments of any of the methods described herein where thecomplement-associated disorder is aHUS, the aHUS can be genetic,acquired, or idiopathic form. In some embodiments, the aHUS can becomplement factor H (CFH)-associated aHUS (e.g., due to mutations in CFHor the presence of antibodies in the subject that bind to CFH), membranecofactor protein (MCP)-associated aHUS, complement factor I(CFI)-associated aHUS, C4b-binding protein (C4BP)-associated aHUS, a vonWillibrand Factor (vWF)-associated disorder, complement factorB-(CFB)-associated aHUS, or a disorder of the alternative pathway thatresults in low C3 levels as a result of increased C3 consumption.

In some embodiments, any of the methods described herein can furtherinclude identifying the subject as one having, suspected of having, orat risk for developing, aHUS.

In some embodiments, any of the methods described herein can include,after the administering, monitoring the subject for an improvement inone or more symptoms of aHUS.

In some embodiments of any of the methods described herein, thecomposition can be administered to the subject prior to, during, orfollowing a plasma therapy (e.g., plasma exchange or plasma infusion).In some embodiments, administration of the C5 inhibitor to the subjectcan alleviate the need for plasma therapy by a patient. For example, insome embodiments, administration (e.g., chronic administration) of theC5 inhibitor to the subject can alleviate or substantially reduce theneed for plasma therapy by a patient for at least 2 months (e.g., 3months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, or 12 months or 1, 2, 3, 4, 5, or 6 years or more).In some embodiments, any of the methods described herein can includeadministering to the subject one or more additional active agents usefulfor treating typical HUS or aHUS. The one or more additional activeagents can be, e.g., selected from the group consisting ofanti-hypertensives, anti-platelet agents, prostacyclin, fibrinolyticagents, and anti-oxidants.

In some embodiments, the human is an infant. The infant can be, e.g.,0.5 (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, or 9.5) years old. The infant can be less than 10 (e.g., lessthan 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2,1.5, 1, or less than 1) year(s) old.

In embodiments of any of the methods described herein where thecomplement-associated disorder is typical HUS, the typical HUS can beassociated with an E. coli infection in or on the human. The E. coliinfection can be, e.g., an E. coli O157 (e.g., O157:H7), O26, O103,O111, or O145 infection. In some embodiments of any of the methodsdescribed herein, the typical hemolytic uremic syndrome can beassociated with a Shigella dysenteriae infection in or on the human. TheShigella dysenteriae infection can be a Shigella dysenteriae type 1infection.

In some embodiments, any of the methods described herein can furtherinclude identifying the human as one having, suspected of having, or atrisk for developing, typical hemolytic uremic syndrome.

In some embodiments, any of the methods described herein can include,after the administering, monitoring the human for an improvement in oneor more symptoms of typical hemolytic uremic syndrome.

In embodiments of any of the methods described herein where thecomplement-associated disorder is CAPS, the CAPS can be associated witha precipitating condition. Precipitating conditions can include, e.g., acancer, transplantation, an infection, surgery, primary antiphospholipidsyndrome, or an autoimmune disorder such as rheumatoid arthritis orsystemic lupus erythematosus. Accordingly, in some embodiments, the CAPScan be associated with a cancer such as, but not limited to, gastriccancer, ovarian cancer, lymphoma, leukemia, endometrial cancer,adenocarcinoma, lung cancer, or any other cancers known in the art toprecipitate or be associated with CAPS. In some embodiments, the CAPScan be idiopathic.

In some embodiments, any of the methods described herein can alsoinclude identifying the human as one having, suspected of having, or atrisk for developing, CAPS. In some embodiments, any of the methodsdescribed herein can include, after the administering, monitoring thehuman for an improvement in one or more symptoms of CAPS.

In some embodiments of any of the methods described herein, thecomposition can be administered to the human prior to, during, orfollowing a plasma exchange, plasmapheresis, IVIG, or any otheradditional therapy for treating CAPS.

In some embodiments, any of the methods described herein can alsoinclude administering to the human one or more additional active agentsuseful for treating CAPS. The one or more additional active agents canbe selected from the group consisting of anti-hypertensives,anti-cytokine agents, steroids, anti-coagulants, or fibrinolytic agents.

In embodiments of any of the methods described herein where thecomplement-associated disorder is TTP, the TTP can be inherited. Forexample, a human can carry one or more (e.g., two, three, four, or fiveor more) mutations in the ADAMTS13 gene. In some embodiments of any ofthe methods described herein, the TTP can be an acquired form. Forexample, in some embodiments, the human can produce antibodies that bindto, and inhibit, the ADAMTS13 metalloproteinase. In some embodiments ofany of the methods described herein, the TTP can be a recurrent form.For example, the human can be one who has had TTP. In some embodimentsof any of the methods described herein, the TTP (or recurrent TTP) isassociated with a precipitating condition such as, but not limited to, acancer, pregnancy, bacterial or viral infection, surgery, or any otherTTP-associated condition known in the art or described herein. In someembodiments of any of the methods described herein, the TTP (orrecurrent TTP) is associated with the use of a therapeutic agentassociated with TTP. For example, the TTP can be associated with the useof, e.g., a platelet aggregation inhibitor such as ticlopidine orclopidogrel or an immunosuppressant (e.g., cyclosporine, mitomycin C,FK506, or interferon-alpha).

In some embodiments, any of the methods described herein can includeidentifying the human as one having, suspected of having, or at risk fordeveloping, TTP. In some embodiments, any of the methods describedherein can include, after the administering, monitoring the human for animprovement in one or more symptoms of TTP.

In some embodiments of any of the methods described herein, thecomposition can be administered to the human prior to, during, orfollowing a plasma exchange, plasma infusion, plasmapheresis, or asplenectomy. In some embodiments, any of the methods described hereincan include administering to the human one or more additional activeagents useful for treating or preventing TTP. The one or more additionalactive agents can be selected from the group consisting ofanti-hypertensives, steroids, anti-coagulants, or fibrinolytic agents.

In embodiments of any of the methods described herein where thecomplement-associated disorder is DDD, the DDD can be an inherited formof the disorder. For example, a human can have a DDD-associated mutationin the complement factor H gene, the complement factor H-related 5 gene,or the complement component C3 gene.

In some embodiments, any of the methods described herein can includeidentifying the human as one having, suspected of having, or at risk fordeveloping, DDD. In some embodiments, any of the methods describedherein can include, after the administering, monitoring the human for animprovement in one or more symptoms of DDD.

In some embodiments of any of the methods described herein, thecomposition can be administered to the human prior to, during, orfollowing a plasma exchange, plasma replacement, plasmapheresis, orintravenous gamma globulin therapy. In some embodiments, any of themethods described herein can include administering to the human one ormore additional active agents useful for treating DDD. The one or moreadditional active agents can be selected from the group consisting ofanti-hypertensives, corticosteroids, anti-coagulants, or fibrinolyticagents.

In embodiments of any of the methods described herein where thecomplement-associated disorder is MG, the human can be one expressing anMG-associated autoantibody such as, but not limited to, an MG-associatedanti-AChR antibody, an MG-associated anti-MuSK antibody, or anMG-associated anti-striational protein antibody. The MG can be ocular MGand/or a drug-induced form of MG such as D-penicillamine-induced MG. Insome embodiments, the human can be in, or be at risk for developing,myasthenic crisis. In some embodiments, the human can be a neonatehaving neonatal MG, wherein a mother with MG passes MG-associatedantibodies through the placenta to an infant.

In some embodiments, any of the methods described herein can furtherinclude identifying the human as one having, suspected of having, or atrisk for developing, MG. In some embodiments, any of the methodsdescribed herein can further include, after the administering,monitoring the human for an improvement in one or more symptoms of MG.In some embodiments of any of the methods described herein, thecomposition can be administered to the human prior to, during, orfollowing a plasma exchange, plasmapheresis, IVIG, or immunoadsorptiontherapy.

In some embodiments, any of the methods described herein can includeadministering to the human one or more additional active agents usefulfor treating or preventing MG. The one or more additional active agentscan be, e.g., acetylcholinesterase inhibitors, immunosuppressive agents,or any other additional active agents useful for treating MG that areknown in the art or described herein.

In embodiments of any of the methods described herein where thecomplement-associated disorder is paroxysmal cold hemoglobinuria (PCH),the PCH can be associated with an infection (e.g., a viral or bacterialinfection) or a neoplasm. For example, the PCH can be associated with aTreponema palladium infection, an influenza virus infection, avaricella-zoster virus infection, a cytomegalovirus (CMV) infection, anEpstein-Barr virus (EBV) infection, an adenovirus infection, aparvovirus B19 infection, a Coxsackie A9 infection, a Haemophilusinfluenza infection, a Mycoplasma pneumoniae infection, or a Klebsiellapneumoniae infection. In some embodiments, the PCH can be associatedwith non-Hodgkin's lymphoma. In some embodiments, the PCH can beassociated with an immunization (e.g., a measles immunization). In someembodiments of any of the methods described herein, the PCH can be acuteor recurrent.

In some embodiments, any of the methods described herein can includeidentifying the human as one having, suspected of having, or at risk fordeveloping, PCH. In some embodiments, any of the methods describedherein can include, after the administering, monitoring the human for animprovement in one or more symptoms of PCH.

In some embodiments of any of the methods described herein, thecomposition can be administered to the human prior to, during, orfollowing a plasma exchange, plasma infusion, IVIG therapy, red celltransfusion, or plasmapheresis. In some embodiments, any of the methodsdescribed herein can include administering to the human one or moreadditional active agents useful for treating or preventing PCH. The oneor more additional active agents can be selected from the groupconsisting of anti-hypertensives, steroids, immunosuppressives (e.g.,rituximab), antibiotics, anti-viral agents, and chemotherapeutic agents.

In embodiments of any of the methods described herein where thecomplement-associated disorder is CAD, the CAD can be associated with aninfection (e.g., a viral or bacterial infection) or a neoplasm. Forexample, the CAD can be associated with an HIV infection, acytomegalovirus (CMV) infection, an Epstein-Barr virus (EBV) infection,or a Mycoplasma pneumoniae infection. In some embodiments, the CAD canbe associated with non-Hodgkin's lymphoma. In some embodiments of any ofthe methods described herein, the CAD can be primary or secondary.

In some embodiments, any of the methods described herein can includeidentifying the human as one having, suspected of having, or at risk fordeveloping, CAD. In some embodiments, any of the methods describedherein can include, after the administering, monitoring the human for animprovement in one or more symptoms of CAD.

In some embodiments of any of the methods described herein, thecomposition can be administered to the human prior to, during, orfollowing a plasma exchange, plasma replacement, IVIG therapy, orplasmapheresis. In some embodiments, any of the methods described hereincan include administering to the human one or more additional activeagents useful for treating or preventing CAD. The one or more additionalactive agents can be selected from the group consisting ofanti-hypertensives, steroids, immunosuppressives (e.g., rituximab),antibiotics, anti-viral agents, and chemotherapeutic agents.

In embodiments of any of the methods described herein where thecomplement-associated disorder is HELLP syndrome, the affected woman canbe pregnant or can be a woman who has recently been pregnant. Forexample, the woman can be one who has given birth less than 14 days(e.g., less than 13 days, 12 days, 11 days, 10 days, nine days, eightdays, seven days, six days, five days, four days, three days, two days,24 hours, 18 hours, 12 hours, 6 hours, or less than 4, 3, 2, or 1 hours)prior to administration. In some embodiments, the woman has beenpregnant more than one time. In some embodiments, the woman can be onewho has developed preeclampsia or HELLP syndrome during at least oneprior pregnancy.

In embodiments where the complement-associated disorder is HELLPsyndrome, the methods described herein can further include the step ofidentifying the woman as one having, suspected of having, or at risk fordeveloping, HELLP syndrome. In some embodiments, any of the methodsdescribed herein can further include the step of, after theadministering, monitoring the woman for an improvement in one or moresymptoms of HELLP syndrome.

In some embodiments of any of the methods described herein, thecomposition can be administered to the woman prior to, during, orfollowing a plasma exchange, plasmapheresis, platelet transfusion, orred blood cell transfusion.

In some embodiments, any of the methods described herein can include thestep of administering to the woman at least one or more additionalactive agents useful for treating or preventing HELLP syndrome in awoman. The one or more additional active agents can be selected from thegroup consisting of an anti-hypertensive, a steroid, an anti-seizureagent, and an anti-thrombotic agent.

In yet another aspect, the disclosure features an article ofmanufacture, which includes (or consists of) a container with a labeland a composition containing an inhibitor of human complement (e.g., aninhibitor of human complement component C5). The label indicates thatthe composition is to be administered to a human having, suspected ofhaving, or at risk for developing, a complement-associated disorder suchas any of the complement-associated disorders described herein. Theinhibitor can be, e.g., an antibody or antigen-binding fragment thereofthat binds to complement component C5 or a fragment thereof such as C5aor C5b. In some embodiments, the article of manufacture contains one ormore additional active agents that are useful for treating or preventinga complement-associated disorder (e.g., ameliorating one or moresymptoms of the disorder).

The disclosure is also based, in part, on the discovery by the inventorsthat upon treatment with the C5 inhibitor eculizumab, a patient with thecomplement-associated disorder aHUS and thrombotic microangiopathy (TMA)in the kidney experienced a complete resolution of the TMA in the kidneywith no further development of TMA. Accordingly, in another aspect, thedisclosure features a method for treating thrombotic microangiopathy(TMA), or reducing the occurrence or severity of TMA, in a patient whohas, is suspected of having, or at risk of developing TMA. The methodincludes administering to the patient (being in need thereof) aninhibitor of complement such as an inhibitor of complement component C5to thereby treat TMA in the patient. The inhibitor can be, e.g., any ofthe C5 inhibitors described herein, e.g., eculizumab. Administration ofthe C5 inhibitor can reduce the occurrence or severity of TMA in thebrain and/or kidney of the patient. In some embodiments, administrationof the C5 inhibitor treats or promotes the resolution of pre-existingTMA in the patient, e.g., a pre-existing TMA in the brain or kidney ofthe patient.

In some embodiments, the patient has a complement associated-disordersuch as any of those described herein, e.g., membranoproliferativeglomerulonephritis, Degos disease, atypical hemolytic uremic syndrome,antibody-mediated rejection, HELLP syndrome, or catastrophicantiphospholipid syndrome.

The inventors have also discovered that administration of eculizumab topatients with, e.g., aHUS or CAPS results in an unexpectedly rapidamelioration of one or more symptoms of the diseases. For example, theinventors have discovered that hypertension, reduced urine output, andlow platelet levels are ameliorated in eculizumab-treated aHUS patientsin less than one month (e.g., less than two weeks) from initiatingchronic treatment with eculizumab. In another example, the inventorsdiscovered that the proteinuria in a patient with membranoproliferativeglomerulonephritis was ameliorated within a month following initiationof chronic treatment with eculizumab. Accordingly, in yet anotheraspect, the disclosure features a method for ameliorating one or moresymptoms associated with a complement-associated disorder such as any ofthe complement-associated disorders described herein with the exceptionof paroxysmal nocturnal hemoglobinuria. The method includesadministering to a patient in need thereof an inhibitor of complement(e.g., an inhibitor of complement component C5) in an amount effectiveto ameliorate one or more symptoms associated a complement-associateddisorder, wherein the symptoms are ameliorated within less than twomonths (e.g., less than 7, 6, 5, 4, 3, or 2 weeks; less than 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s); orless than 12, 11, 10, 9, 8, 7, 6 or even less than 5 hours) afteradministering the inhibitor. Symptoms of complement-associated disordersare well known in the art of medicine and described herein. Thecomplement inhibitor can be any of the C5 inhibitors described herein,e.g., eculizumab. Exemplary symptoms that may be ameliorated by the C5inhibitor in less than 2 months include, e.g., proteinuria,hypertension, reduced platelet counts, and reduced urine output from thekidney. In some embodiments, at least one of the symptoms is amelioratedto within 40 (e.g., 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or even 1) % of its normal level or value. Forexample, in some embodiments, administration of the C5 inhibitoreculizumab to a hypertensive patient with aHUS can ameliorate thepatient's hypertension to within 40% of the normal blood pressure(diastolic and/or systolic) for the patient. In some embodiments,administration of the C5 inhibitor can completely ameliorate one or moresymptoms of the complement-associated disorder in the subject. In someembodiments, the patient has had a kidney transplant, e.g., an aHUSpatient who has recently undergone a kidney transplant. The complementassociated-disorder can be any of those described herein, e.g.,membranoproliferative glomerulonephritis, Degos disease, atypicalhemolytic uremic syndrome, antibody-mediated rejection, HELLP syndrome,and catastrophic antiphospholipid syndrome.

Many of the complement-associated disorders described herein arecharacterized by episodic or sporadic symptom presentation andhistorically have only been treated when symptoms manifest. However, theinventors have discovered that an underlying complement-associateddisorder remains present even when the patients are asymptomatic. Theinventors have also discovered that recurrences or relapses of thedisorders can be prevented or at least minimized by chronic treatmentusing a complement-mediated inhibitor. Such chronic administration ofthe inhibitor is useful to prevent or minimize the often irreversibledamage (e.g., loss of an organ such as a kidney) inflicted upon patientswith severe complement-related disorders (e.g., aHUS or CAPS) when therelapses occur. Accordingly, it is of the utmost importance toadminister a complement inhibitor to the patient in an amount and with afrequency sufficient to continually maintain a concentration of theinhibitor that is high enough to prevent or substantially inhibitsystemic complement activity in the patients.

Thus, in another aspect, the disclosure features a method for treating acomplement-associated disorder, which method includes chronicallyadministering to a patient in need thereof a complement inhibitor (e.g.,a C5 inhibitor such as an anti-C5 antibody) in an amount and with afrequency that are effective to maintain systemic complement inhibitionin the patients with the proviso that the complement-associated disorderis not paroxysmal nocturnal hemoglobinuria.

As used herein, “chronically administered,” “chronic treatment,”“treating chronically,” or similar grammatical variations thereof referto a treatment regimen that is employed to maintain a certain thresholdconcentration of a therapeutic agent in the blood of a patient in orderto completely or substantially suppress systemic complement activity inthe patient over a prolonged period of time. Accordingly, a patientchronically treated with a complement inhibitor can be treated for aperiod of time that is greater than or equal to 2 weeks (e.g., 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years or for the remainderof the patient's life) with the inhibitor in an amount and with a dosingfrequency that are sufficient to maintain a concentration of theinhibitor in the patient's blood that inhibits or substantially inhibitssystemic complement activity in the patient. In some embodiments, thecomplement inhibitor can be chronically administered to a patient inneed thereof in an amount and with a frequency that are effective tomaintain serum hemolytic activity at less than or equal to 20 (e.g., 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or even below 5) % andto maintain serum hemolytic activity at less than or equal to 20%. See,e.g., Hill et al. (2005) Blood 106(7):2559. In some embodiments, thecomplement inhibitor can be administered to a patient in an amount andwith a frequency that are effective to maintain serum lactatedehydrogenase (LDH) levels at within at least 20 (e.g., 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or even below 5) % of the normalrange for LDH. See Hill et al. (2005) supra. In some embodiments, thecomplement inhibitor is administered to the patient in an amount andwith a frequency that are effective to maintain a serum LDH level lessthan 550 (e.g., less than 540, 530, 520, 510, 500, 490, 480, 470, 460,450, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310,300, 290, 280, or less than 270) IU/L. To maintain systemic complementinhibition in a patient, the complement inhibitor can be chronicallyadministered to the patient, e.g., once a week, once every two weeks,twice a week, once a day, once a month, or once every three weeks. Insome embodiments of any of the methods described herein, a C5 inhibitor(e.g., an anti-C5 antibody) can be administered to a patient in anamount and with a frequency of administration effective to maintain aconcentration of at least 0.7 (e.g., at least 0.8, 0.9, one, two, three,four, five, six, seven, eight, nine, or 10 or more) divalent C5inhibitor molecule(s) (e.g., a whole anti-C5 antibody such aseculizumab) per every C5 molecule in the patient's blood. “Divalent” or“bivalent,” with respect to a C5 inhibitor, refers to a C5 inhibitorthat contains at least two binding sites for a C5 molecule. Where the C5inhibitor is monovalent (e.g., a single chain anti-C5 antibody or a Fabthat binds to C5), the inhibitor can be administered to the patient inan amount and with a frequency that are effective to maintain aconcentration of at least 1.5 (e.g., at least 2, 2.5, 3, 3.5, 4, 4.5, or5 or more) of the monovalent C5 inhibitors per every C5 molecule in theblood. In some embodiments, the monovalent C5 inhibitor can beadministered to the patient in an amount and with a frequency that areeffective to maintain a ratio of monovalent C5 inhibitor to C5 of atleast 2:1 (e.g., at least 3:1, at least 4:1, at least 5:1, or at least6:1 or more). In some embodiments of any of the methods describedherein, a whole (bivalent) anti-C5 antibody is administered to thepatient in an amount and with a frequency that are effective to maintaina concentration of at least 40 (e.g., 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, or 120 or more) μg of theantibody per milliliter of the patient's blood. In preferredembodiments, a whole anti-C5 antibody (e.g., eculizumab) is administeredin an amount and with a frequency to maintain the antibody at aconcentration of at least 50 μg per milliliter of the patient's blood.In preferred embodiments, a whole anti-C5 antibody (e.g., eculizumab) isadministered in an amount and with a frequency to maintain the antibodyat a concentration of at least 100 μg per milliliter of the patient'sblood. In some embodiments of any of the methods described herein, amonovalent anti-C5 antibody (e.g., a single chain antibody or an Fabfragment) can be administered to the patient in an amount and with afrequency that are effective to maintain a concentration of at least 80(e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, or a 170 or more) μg of the antibody per milliliter of thepatient's blood. Exemplary chronic dosing strategies are describedherein.

In another aspect, the disclosure features a method for treating acomplement-associated disorder, which method includes chronicallyadministering to a patient in need thereof an anti-C5 antibody in anamount and with a frequency that are effective to maintain systemiccomplement inhibition in the patients. In some embodiments, the anti-C5antibody can be chronically administered to a patient in need thereof inan amount and with a frequency that are effective to maintain serumhemolytic activity at less than or equal to 20 (e.g., 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or even below 5) % and to maintainthe serum hemolytic activity at less than or equal to 20%. See, e.g.,Hill et al. (2005) Blood 106(7):2559. In some embodiments, the anti-C5antibody can be administered to a patient in an amount and with afrequency that are effective to maintain serum lactate dehydrogenase(LDH) levels at within: at least 20 (e.g., 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, or even below 5) % of the normal range for LDH;or less than or equal to 550 (e.g., less than or equal to 550, 540, 530,520, 510, 500, 490, 480, 470, 460, 450, 430, 420, 410, 400, 390, 380,370, 360, 350, 340, 330, 320, 310, 300, 290, 280, or less than 270)IU/L. See, e.g., Hill et al. (2005) supra. In some embodiments, theanti-C5 antibody is administered to the patient in an amount and with afrequency to maintain a concentration of at least 0.7 (e.g., at least0.8, 0.9, 1, 2, 3, or 4 or more) whole (bivalent) anti-C5 antibodymolecule(s) per every C5 molecule in the patient's blood. In someembodiments, the anti-C5 antibody can be administered to the patient inan amount and with a frequency that are effective to maintain a ratio ofwhole (bivalent) anti-C5 antibody to C5 in the blood of at least 1:1(e.g., at least 3:2, 2:1, 5:2, or 3:1). Where the anti-C5 antibody ismonovalent, the anti-C5 antibody can be administered to the patient inan amount and with a frequency that are effective to maintain aconcentration of at least 2 of the monovalent anti-C5 antibodies perevery C5 molecule in the blood. In some embodiments, the monovalentanti-C5 antibody can be administered to the patient in an amount andwith a frequency that are effective to maintain a ratio of monovalentanti-C5 antibody to C5 of at least 2:1 (e.g., at least 3:1, at least4:1, at least 5:1, or 6:1 or more). The anti-C5 antibody can be, e.g.,eculizumab. The patient can have, be suspected of having, or be at riskfor developing a complement-associated disorder with the proviso thatthe disorder is not paroxysmal nocturnal hemoglobinuria. For example,the complement-associated disorder can be one selected from the groupconsisting of membranoproliferative glomerulonephritis, Degos disease,atypical hemolytic uremic syndrome, antibody-mediated rejection, HELLPsyndrome, and catastrophic antiphospholipid syndrome.

In some embodiments of any of the methods described herein, an anti-C5antibody can be administered chronically to a patient based on his orher weight. In some embodiments of any of the methods described herein,an anti-C5 antibody (e.g., eculizumab) can be administered chronicallyto a patient based on his or her weight and under the dosing scheduleset forth in Table 1.

TABLE 1 Exemplary Chronic Dosing Schedules for a Whole Anti-C5 Antibody(e.g., eculizumab) by Patient Weight Induction/Loading MaintenanceDosing Patient Weight Dosing (A) (B) Adults of any At least 800 (e.g.,at At least 800 Following the (A) weight or any least 810, 820, 830,(e.g., at least dose, at least 800 patient with a 840, 850, 860, 870,880, 810, 820, 830, (e.g., at least 810, body weight that 890, 900, 910,920, 930, 840, 850, 860, 820, 830, 840, 850, is greater than or 940,950, 960, 970, 980, 870, 880, 890, 860, 870, 880, 890, equal to 40 kg990, 1000, 1100, or 900, 910, 920, 900, 910, 920, 930, 1200 or more) mgonce 930, 940, 950, 940, 950, 960, 970, a week for four weeks 960, 970,980, 980, 990, 1000, 990, 1000, 1050, 1100, 1150, 1050, 1100, 1200,1250, 1300, 1150, 1200, 1350, or 1400 or 1250, 1300, more) mg once 1350,or 1400 every two weeks or more) mg thereafter* on week five Body weightthat At least 500 (e.g., at At least 800 Following the (A) is less than40 kg, least 510, 520, 530, (e.g., at least dose, at least 800 butgreater than or 540, 550, 560, 570, 580, 810, 820, 830, (e.g., at least810, equal to 30 kg 590, 600, 610, 620, 630, 840, 850, 860, 820, 830,840, 850, 640, 650, 660, 670, 680, 870, 880, 890, 860, 870, 880, 890,690, 700, 710, 720, 730, 900, 910, 920, 900, 910, 920, 930, 740, 750,760, 770, 780, 930, 940, 950, 940, 950, 960, 970, 790, 800, or 850 or960, 970, 980, 980, 990, 1000, more) mg once a week 990, 1000, 1050,1100, 1150, for two weeks 1050, 1100, 1200, 1250, 1300, 1150, 1200,1350, or 1400 or 1250, 1300, more) mg once 1350, or 1400 every two weeksor more) mg thereafter* on week three Body weight that At least 500(e.g., at At least 500 Following the (A) is less than 30 kg, least 510,520, 530, (e.g., at least dose, at least 500 but greater than or 540,550, 560, 570, 580, 510, 520, 530, (e.g., at least 510, equal to 20 kg590, 600, 610, 620, 630, 540, 550, 560, 520, 530, 540, 550, 640, 650,660, 670, 680, 570, 580, 590, 560, 570, 580, 590, 690, 700, 710, 720,730, 600, 610, 620, 600, 610, 620, 630, 740, 750, 760, 770, 780, 630,640, 650, 640, 650, 660, 670, 790, 800, or 850 or 660, 670, 680, 680,690, 700, 710, more) mg once a week 690, 700, 710, 720, 730, 740, 750,for two weeks 720, 730, 740, 760, 770, 780, 790, 750, 760, 770, 800, or850 or more) 780, 790, 800, mg once every two or 850 or weeksthereafter* more) mg on week three Body weight that At least 500 (e.g.,at At least 200 Following the (A) is less than 20 kg, least 510, 520,530, (e.g., at least dose, at least 200 but greater than or 540, 550,560, 570, 580, 210, 220, 230, (e.g., at least 210, equal to 10 kg 590,600, 610, 620, 630, 240, 250, 260, 220, 230, 240, 250, 640, 650, 660,670, 680, 270, 280, 290, 260, 270, 280, 290, 690, 700, 710, 720, 730,300, 310, 320, 300, 310, 320, 330, 740, 750, 760, 770, 780, 330, 340,350, 340, 350, 360, 370, 790, 800, or 850 or 360, 370, 380, 380, 390,400, 410, more) mg once a week 390, 400, 410, 420, 430, 440, 450, forone week 420, 430, 440, 460, 470, 480, 490, 450, 460, 470, 500, or 550or more) 480, 490, 500, mg once every two or 550 or weeks thereafter*more) mg on week two Body weight that At least 200 (e.g., at At least200 Following the (A) is less than 10 kg, least 210, 220, 230, (e.g., atleast dose, at least 200 but greater than or 240, 250, 260, 270, 280,210, 220, 230, (e.g., at least 210, equal to 5 kg 290, 300, 310, 320,330, 240, 250, 260, 220, 230, 240, 250, 340, 350, 360, 370, 380, 270,280, 290, 260, 270, 280, 290, 390, 400, 410, 420, 430, 300, 310, 320,300, 310, 320, 330, 440, 450, 460, 470, 480, 330, 340, 350, 340, 350,360, 370, 490, 500, or 550 or 360, 370, 380, 380, 390, 400, 410, more)mg once a week 390, 400, 410, 420, 430, 440, 450, for one week 420, 430,440, 460, 470, 480, 490, 450, 460, 470, 500, or 550 or more) 480, 490,500, mg once every three or 550 or weeks thereafter* more) mg on weektwo *In accordance with the present disclosure, the (B) maintenancedosing schedule can be maintained for the duration of the treatmentregimen, e.g., at least one (e.g., at least two, three, four, five, six,seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24,36, or 48 or more) month(s); at least one (e.g., at least two, three,four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, or 15 or more)years; or for the remainder of the patient's life.

In preferred embodiments, an anti-C5 antibody (e.g., eculizumab) can beadministered to a patient based on his or her weight under the dosingschedules set forth in Table 2.

TABLE 2 Exemplary Chronic Dosing Schedules for a Whole Anti-C5 Antibody(e.g., eculizumab) by Patient Weight Induction/Loading MaintenanceDosing Patient Weight Dosing (A) (B) Adults of any At least 900 (e.g.,at At least 1200 Following the (A) weight or any least 910, 920, 930,(e.g., at least dose, at least 1200 patient with a 940, 950, 960, 970,980, 1225, 1250, (e.g., at least 1225, body weight that 990, 1000, 1100,or 1300, 1350, or 1250, 1300, 1350, or is greater than or 1200 or more)mg once 1400 or more) 1400 or more) mg equal to 40 kg a week for fourweeks mg on week once every two five weeks thereafter* Body weight thatAt least 600 (e.g., at At least 900 Following the (A) is less than 40kg, least 610, 620, 630, (e.g., at least dose, at least 900 but greaterthan or 640, 650, 660, 670, 680, 910, 920, 930, (e.g., at least 910,equal to 30 kg 690, 700, 710, 720, 730, 940, 950, 960, 920, 930, 940,950, 740, 750, 760, 770, 780, 970, 980, 990, 960, 970, 980, 990, 790,800, or 850 or 1000, 1050, 1000, 1050, 1100, more) mg once a week 1100,1150, or 1150, or 1200 or for two weeks 1200 or more) more) mg once mgon week every two weeks three thereafter* Body weight that At least 600(e.g., at At least 600 Following the (A) is less than 30 kg, least 610,620, 630, (e.g., at least dose, at least 600 but greater than or 640,650, 660, 670, 680, 610, 620, 630, (e.g., at least 610, equal to 20 kg690, 700, 710, 720, 730, 640, 650, 660, 620, 630, 640, 650, 740, 750,760, 770, 780, 670, 680, 690, 660, 670, 680, 690, 790, 800, or 850 or700, 710, 720, 700, 710, 720, 730, more) mg once a week 730, 740, 750,740, 750, 760, 770, for two weeks 760, 770, 780, 780, 790, 800, or 790,800, or 850 or more) mg 850 or more) once every two mg on week weeksthereafter* three Body weight that At least 600 (e.g., at At least 300Following the (A) is less than 20 kg, least 610, 620, 630, (e.g., atleast dose, at least 300 but greater than or 640, 650, 660, 670, 680,310, 320, 330, (e.g., at least 310, equal to 10 kg 690, 700, 710, 720,730, 340, 350, 360, 320, 330, 340, 350, 740, 750, 760, 770, 780, 370,380, 390, 360, 370, 380, 390, 790, 800, or 850 or 400, 410, 420, 400,410, 420, 430, more) mg once a week 430, 440, 450, 440, 450, 460, 470,for one week 460, 470, 480, 480, 490, 500, or 490, 500, or 550 or more)mg 550 or more) once every two mg on week weeks thereafter* two Bodyweight that At least 300 (e.g., at At least 300 Following the (A) isless than 10 kg, least 310, 320, 330, (e.g., at least dose, at least 300but greater than or 340, 350, 360, 370, 380, 310, 320, 330, (e.g., atleast 310, equal to 5 kg 390, 400, 410, 420, 430, 340, 350, 360, 320,330, 340, 350, 440, 450, 460, 470, 480, 370, 380, 390, 360, 370, 380,390, 490, 500, or 550 or 400, 410, 420, 400, 410, 420, 430, more) mgonce a week 430, 440, 450, 440, 450, 460, 470, for one week 460, 470,480, 480, 490, 500, or 490, 500, or 550 or more) mg 550 or more) onceevery three mg on week weeks thereafter* two *In accordance with thepresent disclosure, the (B) maintenance dosing schedule can bemaintained for the duration of the treatment regimen, e.g., at least one(e.g., at least two, three, four, five, six, seven, eight, nine, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 36, or 48 or more) month(s);at least one (e.g., at least two, three, four, five, six, seven, eight,nine, 10, 11, 12, 13, 14, or 15 or more) years; or for the remainder ofthe patient's life.It is understood that the exemplary dosing schedules in Tables 1 or 2can be adjusted (in frequency, duration, and/or in total amount ofantibody administered) by a medical practitioner as necessary in such away as to maintain complete or substantially complete inhibition ofsystemic complement activity in the patient for the duration of thedosing regime.

In another aspect, the disclosure features a method for treating acomplement-associated disorder, the method including chronicallyadministering to a patient in need thereof an anti-C5 antibody in anamount and with a frequency that are effective to maintain aconcentration of at least 40 (e.g., at least 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, or 120 or more) μg ofthe antibody per milliliter of the patient's blood, wherein the patienthas, is suspected of having, or is at risk for developing acomplement-associated disorder with the proviso that the disorder is notparoxysmal nocturnal hemoglobinuria.

In some embodiments, the anti-C5 antibody is administered to the patientat least once every two weeks. In some embodiments, the anti-C5 antibodyis administered to the patient once per week. In some embodiments, theanti-C5 antibody is administered to the patient for at least 9 weeks(e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years or for the remainderof the patient's life) under the following dosing schedule: at least 800(e.g., at least 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, or 1200 or more) mgof the anti-C5 antibody, once per week for four consecutive weeks; atleast 800 (e.g., at least 810, 820, 830, 840, 850, 860, 870, 880, 890,900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, or 1200 ormore) mg of the anti-C5 antibody once during the fifth week; and atleast 800 (e.g., at least 810, 820, 830, 840, 850, 860, 870, 880, 890,900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, or 1200 ormore) mg of the anti-C5 antibody bi-weekly thereafter for the remainderof the dosing schedule. In preferred embodiments, at least 900 mg of theanti-C5 antibody is administered to the patient, once per week for fourweeks; at least 1200 mg is administered to the patient during the fifthweek; and at least 1200 mg of the anti-C5 antibody is administered tothe patient bi-weekly thereafter for the remainder of the chronic dosingschedule.

In yet another aspect, the disclosure features a method fortransplanting an organ or tissue. The method includes transplanting anorgan or tissue into a patient in need thereof, wherein prior to andchronically following the transplanting an inhibitor of human complementis administered to the patient in an amount and with a frequencyeffective to substantially inhibit systemic complement activity in thepatient. The complement inhibitor can be, e.g., a C5 inhibitor such asan anti-C5 antibody (e.g., eculizumab). As described herein, the C5inhibitor (e.g., the anti-C5 antibody) can be administered in an amountand with a frequency to maintain a concentration of at least 0.7bivalent C5 inhibitor molecule(s) (or at least 1.5 monovalent C5inhibitor molecule(s)) per every C5 molecule in the patient's blood. Insome embodiments, the C5 inhibitor (e.g., the anti-C5 antibody) can beadministered to the patient in an amount and with a frequency tomaintain a concentration of at least at least 40 (e.g., 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, or 120 ormore) μg of the inhibitor (e.g., the anti-C5 antibody) in the patient'sblood. In some embodiments, at least 800 (e.g., at least 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, 1100, or 1200 or more) mg of the anti-C5 antibody (e.g.,eculizumab) is administered to the patient less than 24 (e.g., less than23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, or less than 2) hours prior to transplanting the organ or tissueto the patient. In some embodiments, the methods can also include, priorto the transplanting, contacting (e.g., soaking) the organ or tissuewith a C5 inhibitor (e.g., an anti-C5 antibody such as eculizumab) foran amount of time and under conditions that inhibit complementactivation in the organ or tissue upon transplantation. The organ canbe, e.g., skin, a kidney, heart, lung, limb (e.g., finger or toe), eye,stem cell population, bone marrow, vascular tissue, muscle, nervoustissue, or liver. The patient can have, be at risk for developing, or besuspected of having aHUS. In some embodiments, at least 700 (e.g., atleast 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, 1100, or 1200 or more) mg of an anti-C5 antibody isadministered to the patient less than 24 (e.g., less than 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or lessthan 2) hours following the transplanting. In some embodiments, theanti-C5 antibody is chronically administered to the patient followingthe transplanting. For example, an anti-C5 antibody can be chronicallyadministered to the patient for at least 9 weeks (e.g., 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, or 52 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 10.5, or 12 years or for the remainder of the patient'slife) under the following dosing schedule: at least 700 (e.g., at least710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,990, 1000, 1100, or 1200 or more) mg of the anti-C5 antibody less than24 hours after transplanting the organ or tissue; at least 700 (e.g., atleast 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, 1100, or 1200 or more) mg of the anti-C5 antibody onceper week for four weeks after the initial post-transplant dose; at least700 (e.g., at least 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,950, 960, 970, 980, 990, 1000, 1100, or 1200 or more) mg of the anti-C5antibody once during the fifth week; and at least 700 (e.g., at least710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,990, 1000, 1100, or 1200 or more) mg of the anti-C5 antibody bi-weeklythereafter for the remainder of the dosing schedule. In preferredembodiments, an anti-C5 antibody is administered to a patient undergoinga transplant operation under the following dosing schedule: at least1200 mg of the anti-C5 antibody is administered to the patient less than24 hours prior to the transplanting; at least 900 mg of the anti-C5antibody is administered to the patient within 24 hours after thetransplanting; at least 1200 mg of the anti-C5 antibody is administeredto the patient once a week for four weeks following the firstpost-operation administration of the anti-C5 antibody; 1200 mgadministered to the patient on the fifth week following the firstpost-operation administration of the anti-C5 antibody; and at least 1200mg of the anti-C5 antibody administered to the patient bi-weeklythereafter for the remainder of the chronic treatment regimen.

In some embodiments, the methods can further include administering tothe patient one or more immunosuppressive agents such as, but notlimited to, rapamycin, cyclosporine A, an anti-IL-2 agent, OKT3, andtacrolimus. The one or more immunosuppressive agents can be administeredprior to, during, or following the transplanting. The one or moreimmunosuppressive agents can also be administered before, concurrentlywith, or following administration of the C5 inhibitor.

The disclosure also features a method for reducing complement-mediatedinjury to an organ or a tissue when transplanted into a patient. Themethod includes, prior to transplanting an organ or tissue to a patientin need thereof, contacting the organ or tissue with a pharmaceuticalsolution comprising an inhibitor of C5 for a period of time and underconditions which reduce complement-mediated injury to the organ ortissue when transplanted into the patient. The C5 inhibitor can also beadministered to the patient prior to, during, and/or after thetransplanting of the organ or tissue. The solution can also contain oneor more immunosuppressive agents such as, but not limited to, rapamycin,cyclosporine A, an anti-IL-2 agent, OKT3, and tacrolimus.

The inventors have also discovered that in patients who have had severecomplement-associated disorders such as CAPS and APS and enteredremission, there still remains in the patients a low level of complementactivity that predisposes the patients for relapse or recurrence. Asnoted above, recurrence of symptoms in patients who have had thesesevere disorders can present immediate and sometimes irreversible injuryto major organs such as the kidney. Thus, while the disclosure is in noway limited by one particular theory or mechanism of action, theinventors assert that in order to prevent sudden relapse or recurrence,a patient with a complement-associated disorder (e.g., aHUS and CAPS)should be chronically treated with a C5 inhibitor even after one or moresymptoms of the disorder have been ameliorated and/or even after thepatient enters a clinical remission. Thus, in yet another aspect, thedisclosure features a method for treating a complement-associateddisorder with the proviso that the disorder is not paroxysmal nocturnalhemoglobinuria. The method includes administering to a patient afflictedwith a complement-associated disorder a C5 inhibitor (e.g., an anti-C5antibody such as eculizumab) in an amount effective to treat thecomplement-associated disorder. The C5 inhibitor is administered to thepatient even after one or more (e.g., two, three, four, five, or six ormore) symptoms of the disorder have been ameliorated. In someembodiments, the C5 inhibitor is administered to the patient even afterone or more symptoms have been completely ameliorated. In someembodiments, the C5-inhibitor is administered to the patient even afterthe patient has entered clinical remission. The C5 inhibitor can beadministered, e.g., chronically administered, to the patient for atleast 8 weeks (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years or for theremainder of the patient's life) after one or more symptoms have beenameliorated in the patient and/or the patient enters clinical remission.The complement-associated disorder can be any of those described herein,e.g., membranoproliferative glomerulonephritis, Degos disease, atypicalhemolytic uremic syndrome, antibody-mediated rejection, HELLP syndrome,and catastrophic antiphospholipid syndrome.

While the disclosure is in no way limited by a particular theory ormechanism of action, based on the observations of the effect ofeculizumab in, e.g., aHUS patients, the inventors have concluded thatthe biological activity of complement component C5a may substantiallycontribute to the vasoconstriction and hypertension associated withaHUS. Accordingly, inhibition of C5a using a C5a inhibitor is useful fortreating aHUS and/or ameliorating the vasoconstriction and hypertensionassociated with aHUS. The method includes administering to a patient inneed thereof an inhibitor of complement component C5a in an amounteffective to treat aHUS in the patient. In some embodiments, thevasoconstriction and hypertension associated with aHUS can beameliorated within less than two months (e.g., less than 7, 6, 5, 4, 3,or 2 weeks; less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1day(s); or less than 12, 11, 10, 9, 8, 7, 6 or even less than 5 hours)after administering the C5a inhibitor. In some embodiments, the C5ainhibitor is an antibody (or antigen-binding fragment thereof) thatbinds to a human C5a protein or a fragment thereof having an amino acidsequence that contains, or consists of, at least four (e.g., at leastfour, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, or 17or more) consecutive amino acids depicted in any one of SEQ IDNOs:12-25. In some embodiments, the inhibitor is an antibody that bindsto human C5a protein having the amino acid sequence depicted in SEQ IDNO:12. The C5a inhibitor does not inhibit the cleavage of C5 intofragment C5a and C5b. The C5a inhibitor also does not inhibit C5b or theformation of the membrane attack complex. As described herein, in someembodiments, the C5a inhibitor (e.g., an anti-C5a antibody) can inhibitthe interaction between C5a and a C5a receptor (e.g., C5aR or C5L2). Insome embodiments, the antibody can be a monoclonal antibody, asingle-chain antibody, a humanized antibody, a fully human antibody, apolyclonal antibody, a recombinant antibody, a diabody, a chimerized orchimeric antibody, a deimmunized human antibody, a fully human antibody,a single chain antibody, an Fv fragment, an Fd fragment, an Fabfragment, an Fab′ fragment, or an F(ab′)₂ fragment.

In embodiments of any of the methods described herein where thecomplement-associated disorder is aHUS, the aHUS can be genetic,acquired, or idiopathic form. In some embodiments, the aHUS can becomplement factor H (CFH)-associated aHUS (e.g., aHUS associated withmutations in factor H or autoantibodies that bind to and inactivatefactor H), membrane cofactor protein (MCP)-associated aHUS, complementfactor I (CFI)-associated aHUS, C4b-binding protein (C4BP)-associatedaHUS, complement factor B-(CFB)-associated aHUS, a vWF disorder, or aHUSassociated with any other mutations in the alternative pathway ofcomplement activation causing low levels of C3 as a result of increasedC3 consumption.

In some embodiments, any of the methods described herein can furtherinclude identifying the patient as one having, suspected of having, orat risk for developing, aHUS. In some embodiments, any of the methodsdescribed herein can include, after the administering, monitoring thepatient for an improvement in one or more symptoms of aHUS. In someembodiments of any of the methods described herein, the C5a inhibitorcan be administered to the patient prior to, during, or following aplasma therapy (e.g., plasma exchange or plasma infusion). In someembodiments, administration of the C5a inhibitor to the patient canalleviate the need for plasma therapy by a patient. For example, in someembodiments, administration (e.g., chronic administration) of the C5ainhibitor to the patient can alleviate or substantially reduce the needfor plasma therapy by a patient for at least 2 months (e.g., 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, or 12 months or 1, 2, 3, 4, 5, or 6 years or more). In someembodiments, any of the methods described herein can includeadministering to the patient one or more additional active agents usefulfor treating typical HUS or aHUS. The one or more additional activeagents can be, e.g., selected from the group consisting ofanti-hypertensives, anti-platelet agents, prostacyclin, fibrinolyticagents, and anti-oxidants.

In some embodiments, the human is an infant. The infant can be, e.g.,0.5 (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, or 9.5) years old. The infant can be less than 10 (e.g., lessthan 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2,1.5, 1, or less than 1) year(s) old.

In some embodiments of any of the methods described herein, thecomplement-associated disorder is not paroxysmal nocturnalhemoglobinuria.

“Polypeptide,” “peptide,” and “protein” are used interchangeably andmean any peptide-linked chain of amino acids, regardless of length orpost-translational modification. The complement component C5 proteinsdescribed herein can contain or be wild-type proteins or can be variantsthat have not more than 50 (e.g., not more than one, two, three, four,five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50)conservative amino acid substitutions. Conservative substitutionstypically include substitutions within the following groups: glycine andalanine; valine, isoleucine, and leucine; aspartic acid and glutamicacid; asparagine, glutamine, serine and threonine; lysine, histidine andarginine; and phenylalanine and tyrosine.

The human complement component C5 proteins described herein also include“antigenic peptide fragments” of the proteins, which are shorter thanfull-length and/or immature (pre-pro) C5 proteins, but retain at least10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, at least 99%, at least 99.5%, or 100% or more) of the abilityof the full-length protein to induce an antigenic response in a mammal(see below under “Methods for Producing an Antibody”). Antigenic peptidefragments of a C5 protein include terminal as well internal deletionvariants of the protein. Deletion variants can lack one, two, three,four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 amino acid segments (of two or more amino acids) ornon-contiguous single amino acids. Antigenic peptide fragments can be atleast 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,250, 300, 350, 400, 450, 500, or 600 or more) amino acid residues inlength (e.g., at least 6 contiguous amino acid residues in any one ofSEQ ID NOS:1-11). In some embodiments, an antigenic peptide fragment ofa human C5 protein has fewer than 500 (e.g., fewer than 450, 400, 350,325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120,110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45,44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, or 6) amino acid residues in length (e.g., fewer than 500contiguous amino acid residues in any one of SEQ ID NOS:1-11). In someembodiments, an antigenic peptide fragment of a full-length, immaturehuman C5 protein (prepro-C5 protein) has at least 6, but fewer than 500,amino acid residues in length.

In some embodiments, the human complement component C5 protein can havean amino acid sequence that is, or is greater than, 70 (e.g., 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) % identical to the human C5protein having the amino acid sequence depicted in SEQ ID NO:1 (seebelow).

Percent (%) amino acid sequence identity is defined as the percentage ofamino acids in a candidate sequence that are identical to the aminoacids in a reference sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Alignment for purposes of determining percent sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full-length of thesequences being compared can be determined by known methods.

Amino acid sequences for exemplary human C5 proteins as well asantigenic peptide fragments thereof are known in the art and are setforth below.

As used throughout the present disclosure, the term “antibody” refers toa whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) moleculethat is generated by any one of a variety of methods that are known inthe art and described herein. The term “antibody” includes a polyclonalantibody, a monoclonal antibody, a chimerized or chimeric antibody, ahumanized antibody, a deimmunized human antibody, and a fully humanantibody. The antibody can be made in or derived from any of a varietyof species, e.g., mammals such as humans, non-human primates (e.g.,monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats,dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Theantibody can be a purified or a recombinant antibody.

As used herein, the term “antibody fragment,” “antigen-bindingfragment,” or similar terms refer to fragment of an antibody thatretains the ability to bind to an antigen (e.g., a complement componentC5 protein), e.g., a single chain antibody, a single chain Fv fragment(scFv), an Fd fragment, an Fab fragment, an Fab′ fragment, or an F(ab′)₂fragment. An scFv fragment is a single polypeptide chain that includesboth the heavy and light chain variable regions of the antibody fromwhich the scFv is derived. In addition, diabodies (Poljak (1994)Structure 2(12):1121-1123; Hudson et al. (1999) J. Immunol. Methods23(1-2):177-189, the disclosures of each of which are incorporatedherein by reference in their entirety) and intrabodies (Huston et al.(2001) Hum. Antibodies 10(3-4):127-142; Wheeler et al. (2003) Mol Ther8(3):355-366; Stocks (2004) Drug Discov. Today 9(22): 960-966, thedisclosures of each of which are incorporated herein by reference intheir entirety) that bind to a complement component C5 protein can beincorporated into the compositions, and used in the methods, describedherein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the presently disclosed methods and compositions.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methodsfor treating or preventing a complement-associated disorder, will beapparent from the following description, the examples, and from theclaims.

DETAILED DESCRIPTION

The present disclosure provides compositions containing an inhibitor ofhuman complement component C5 (e.g., an antibody that binds to a humancomplement component C5 protein or a biologically-active fragmentthereof such as C5a and C5b) and methods for using the compositions totreat or prevent a complement-mediated disorder in a human. While in noway intended to be limiting, exemplary compositions (e.g.,pharmaceutical compositions and formulations) and methods for using thecompositions are elaborated on below.

Compositions

The compositions described herein contain an inhibitor of humancomplement. Any compound which binds to or otherwise blocks thegeneration and/or activity of any of the human complement components maybe utilized in accordance with the present disclosure. For example, aninhibitor of complement can be, e.g., a small molecule, a nucleic acidor nucleic acid analog, a peptidomimetic, or a macromolecule that is nota nucleic acid or a protein. These agents include, but are not limitedto, small organic molecules, RNA aptamers, L-RNA aptamers, Spiegelmers,antisense compounds, double stranded RNA, small interfering RNA, lockednucleic acid inhibitors, and peptide nucleic acid inhibitors. In someembodiments, a complement inhibitor may be a protein or proteinfragment.

In some embodiments, the compositions contain antibodies specific to ahuman complement component. Some compounds include antibodies directedagainst complement components C1, C2, C3, C4, C5 (or a fragment thereof;see below), C6, C7, C8, C9, Factor D, Factor B, Factor P, MBL, MASP-1,and MASP-2, thus preventing the generation of the anaphylatoxic activityassociated with C5a and/or preventing the assembly of the membraneattack complex associated with C5b.

The compositions can also contain naturally occurring or soluble formsof complement inhibitory compounds such as CR1, LEX-CR1, MCP, DAF, CD59,Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH. Othercompounds which may be utilized to bind to or otherwise block thegeneration and/or activity of any of the human complement componentsinclude, but are not limited to, proteins, protein fragments, peptides,small molecules, RNA aptamers including ARC187 (which is commerciallyavailable from Archemix Corporation, Cambridge, Mass.), L-RNA aptamers,spiegelmers, antisense compounds, serine protease inhibitors, moleculeswhich may be utilized in RNA interference (RNAi) such as double strandedRNA including small interfering RNA (siRNA), locked nucleic acid (LNA)inhibitors, peptide nucleic acid (PNA) inhibitors, etc.

In some embodiments, the complement inhibitor inhibits the activation ofcomplement. For example, the complement inhibitor can bind to andinhibit the complement activation activity of C1 (e.g., C1q, C1r, orC1s) or the complement inhibitor can bind to and inhibit (e.g., inhibitcleavage of) C2, C3, or C4. In some embodiments, the inhibitor inhibitsformation or assembly of the C3 convertase and/or C5 convertase of thealternative and/or classical pathways of complement. In someembodiments, the complement inhibitor inhibits terminal complementformation, e.g., formation of the C5b-9 membrane attack complex. Forexample, an antibody complement inhibitor may include an anti-C5antibody. Such anti-C5 antibodies may directly interact with C5 and/orC5b, so as to inhibit the formation of and/or physiologic function ofC5b.

In some embodiments, the compositions described herein can contain aninhibitor of human complement component C5 (e.g., an antibody, orantigen-binding fragment thereof, that binds to a human complementcomponent C5 protein or a biologically-active fragment thereof such asC5a or C5b). As used herein, an “inhibitor of complement component C5”is any agent that inhibits: (i) the expression, or proper intracellulartrafficking or secretion by a cell, of a complement component C5protein; (ii) the activity of C5 cleavage fragments C5a or C5b (e.g.,the binding of C5a to its cognate cellular receptors or the binding ofC5b to C6 and/or other components of the terminal complement complex;see above); (iii) the cleavage of a human C5 protein to form C5a andC5b; or (iv) the proper intracellular trafficking of, or secretion by acell, of a complement component C5 protein. Inhibition of complementcomponent C5 protein expression includes: inhibition of transcription ofa gene encoding a human C5 protein; increased degradation of an mRNAencoding a human C5 protein; inhibition of translation of an mRNAencoding a human C5 protein; increased degradation of a human C5protein; inhibition of proper processing of a pre-pro human C5 protein;or inhibition of proper trafficking or secretion by a cell of a human C5protein. Methods for determining whether a candidate agent is aninhibitor of human complement component C5 are known in the art anddescribed herein.

An inhibitor of human complement component C5 can be, e.g., a smallmolecule, a polypeptide, a polypeptide analog, a nucleic acid, or anucleic acid analog.

“Small molecule” as used herein, is meant to refer to an agent, whichhas a molecular weight of less than about 6 kDa and most preferably lessthan about 2.5 kDa. Many pharmaceutical companies have extensivelibraries of chemical and/or biological mixtures comprising arrays ofsmall molecules, often fungal, bacterial, or algal extracts, which canbe screened with any of the assays of the application. This applicationcontemplates using, among other things, small chemical libraries,peptide libraries, or collections of natural products. Tan et al.described a library with over two million synthetic compounds that iscompatible with miniaturized cell-based assays (J. Am. Chem. Soc. (1998)120:8565-8566). It is within the scope of this application that such alibrary may be used to screen for inhibitors of human complementcomponent C5. There are numerous commercially available compoundlibraries, such as the Chembridge DIVERSet. Libraries are also availablefrom academic investigators, such as the Diversity set from the NCIdevelopmental therapeutics program. Rational drug design may also beemployed. For example, rational drug design can employ the use ofcrystal or solution structural information on the human complementcomponent C5 protein. See, e.g., the structures described in Hagemann etal. (2008) J Biol Chem 283(12):7763-75 and Zuiderweg et al. (1989)Biochemistry 28(1):172-85. Rational drug design can also be achievedbased on known compounds, e.g., a known inhibitor of C5 (e.g., anantibody, or antigen-binding fragment thereof, that binds to a humancomplement component C5 protein).

Peptidomimetics can be compounds in which at least a portion of asubject polypeptide is modified, and the three dimensional structure ofthe peptidomimetic remains substantially the same as that of the subjectpolypeptide. Peptidomimetics may be analogues of a subject polypeptideof the disclosure that are, themselves, polypeptides containing one ormore substitutions or other modifications within the subject polypeptidesequence. Alternatively, at least a portion of the subject polypeptidesequence may be replaced with a non-peptide structure, such that thethree-dimensional structure of the subject polypeptide is substantiallyretained. In other words, one, two or three amino acid residues withinthe subject polypeptide sequence may be replaced by a non-peptidestructure. In addition, other peptide portions of the subjectpolypeptide may, but need not, be replaced with a non-peptide structure.Peptidomimetics (both peptide and non-peptidyl analogues) may haveimproved properties (e.g., decreased proteolysis, increased retention orincreased bioavailability). Peptidomimetics generally have improved oralavailability, which makes them especially suited to treatment ofdisorders in a human or animal. It should be noted that peptidomimeticsmay or may not have similar two-dimensional chemical structures, butshare common three-dimensional structural features and geometry. Eachpeptidomimetic may further have one or more unique additional bindingelements.

Nucleic acid inhibitors can be used to decrease expression of anendogenous gene encoding human complement component C5. The nucleic acidantagonist can be, e.g., an siRNA, a dsRNA, a ribozyme, a triple-helixformer, an aptamer, or an antisense nucleic acid. siRNAs are smalldouble stranded RNAs (dsRNAs) that optionally include overhangs. Forexample, the duplex region of an siRNA is about 18 to 25 nucleotides inlength, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length. ThesiRNA sequences can be, in some embodiments, exactly complementary tothe target mRNA. dsRNAs and siRNAs in particular can be used to silencegene expression in mammalian cells (e.g., human cells). See, e.g.,Clemens et al. (2000) Proc. Natl. Acad. Sci. USA 97:6499-6503; Billy etal. (2001) Proc. Natl. Acad. Sci. USA 98:14428-14433; Elbashir et al.(2001) Nature 411:494-8; Yang et al. (2002) Proc. Natl. Acad. Sci. USA99:9942-9947, and U.S. Patent Application Publication Nos. 20030166282,20030143204, 20040038278, and 20030224432. Anti-sense agents caninclude, for example, from about 8 to about 80 nucleobases (i.e. fromabout 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases,or about 12 to about 30 nucleobases. Anti-sense compounds includeribozymes, external guide sequence (EGS) oligonucleotides (oligozymes),and other short catalytic RNAs or catalytic oligonucleotides whichhybridize to the target nucleic acid and modulate its expression.Anti-sense compounds can include a stretch of at least eight consecutivenucleobases that are complementary to a sequence in the target gene. Anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment or, in the case of in vitro assays,under conditions in which the assays are conducted. Hybridization ofantisense oligonucleotides with mRNA (e.g., an mRNA encoding a human C5protein) can interfere with one or more of the normal functions of mRNA.The functions of mRNA to be interfered with include all key functionssuch as, for example, translocation of the RNA to the site of proteintranslation, translation of protein from the RNA, splicing of the RNA toyield one or more mRNA species, and catalytic activity which may beengaged in by the RNA. Binding of specific protein(s) to the RNA mayalso be interfered with by antisense oligonucleotide hybridization tothe RNA. Exemplary antisense compounds include DNA or RNA sequences thatspecifically hybridize to the target nucleic acid, e.g., the mRNAencoding a human complement component C5 protein. The complementaryregion can extend for between about 8 to about 80 nucleobases. Thecompounds can include one or more modified nucleobases.

Modified nucleobases may include, e.g., 5-substituted pyrimidines suchas 5-iodouracil, 5-iodocytosine, and C₅-propynyl pyrimidines such asC₅-propynylcytosine and C₅-propynyluracil. Other suitable modifiednucleobases include, e.g., 7-substituted-8-aza-7-deazapurines and7-substituted-7-deazapurines such as, for example,7-iodo-7-deazapurines, 7-cyano-7-deazapurines,7-aminocarbonyl-7-deazapurines. Examples of these include6-amino-7-iodo-7-deazapurines, 6-amino-7-cyano-7-deazapurines,6-amino-7-aminocarbonyl-7-deazapurines,2-amino-6-hydroxy-7-iodo-7-deazapurines,2-amino-6-hydroxy-7-cyano-7-deazapurines, and2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. See, e.g., U.S. Pat.Nos. 4,987,071; 5,116,742; and 5,093,246; “Antisense RNA and DNA,” D. A.Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1988); Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C.(1991) Anticancer Drug D 6:569-84; Helene (1992) Ann. NY. Acad. Sci.660:27-36; and Maher (1992) Bioassays 14:807-15.

Aptamers are short oligonucleotide sequences that can be used torecognize and specifically bind almost any molecule, including cellsurface proteins. The systematic evolution of ligands by exponentialenrichment (SELEX) process is powerful and can be used to readilyidentify such aptamers. Aptamers can be made for a wide range ofproteins of importance for therapy and diagnostics, such as growthfactors and cell surface antigens. These oligonucleotides bind theirtargets with similar affinities and specificities as antibodies do (see,e.g., Ulrich (2006) Handb Exp Pharmacol. 173:305-326).

In some embodiments, the inhibitor of human C5 is an antibody, orantigen-binding fragment thereof, which binds to a human complementcomponent C5 protein. (Hereinafter, the antibody may sometimes bereferred to as an “anti-C5 antibody.”)

In some embodiments, the anti-C5 antibody binds to an epitope in thehuman pro-C5 precursor protein. For example, the anti-C5 antibody canbind to an epitope in the human complement component C5 proteincomprising, or consisting of, the amino acid sequence depicted in SEQ IDNO:1 (NCBI Accession No. AAA51925 and Haviland et al., supra).

An “epitope” refers to the site on a protein (e.g., a human complementcomponent C5 protein) that is bound by an antibody. “Overlappingepitopes” include at least one (e.g., two, three, four, five, or six)common amino acid residue(s).

In some embodiments, the anti-C5 antibody binds to an epitope in thehuman pro-C5 precursor protein lacking the leader sequence. For example,the anti-C5 antibody can bind to an epitope in the human complementcomponent C5 protein comprising, or consisting of, the amino acidsequence depicted in SEQ ID NO:2, which is a human C5 protein lackingthe amino terminal leader sequence.

In some embodiments, the anti-C5 antibody can bind to an epitope in thealpha chain of the human complement component C5 protein. For example,the anti-C5 antibody can bind to an epitope within, or overlapping with,a protein having the amino acid sequence depicted in SEQ ID NO:3, whichis the human complement component C5 alpha chain protein. Antibodiesthat bind to the alpha chain of C5 are described in, for example, Ameset al. (1994) J Immunol 152:4572-4581.

In some embodiments, the anti-C5 antibody can bind to an epitope in thebeta chain of the human complement component C5 protein. For example,the anti-C5 antibody can bind to an epitope within, or overlapping with,a protein having the amino acid sequence depicted in SEQ ID NO:4, whichis the human complement component C5 beta chain protein. Antibodies thatbind to the C5 beta chain are described in, e.g., Moongkarndi et al.(1982) Immunobiol. 162:397; Moongkarndi et al. (1983) Immunobiol.165:323; and Mollnes et al. (1988) Scand. J. Immunol. 28:307-312.

In some embodiments, the anti-C5 antibody can bind to an epitope within,or overlapping with, an antigenic peptide fragment of a human complementcomponent C5 protein. For example, the anti-C5 antibody can bind to anepitope within, or overlapping with, an antigen peptide fragment of ahuman complement component C5 protein, the fragment containing, orconsisting of, the following amino acid sequence:

(SEQ ID NO: 5) VIDHQGTKSSKCVRQKVEGSS or  (SEQ ID NO: 6) KSSKC.

In some embodiments, the anti-C5 antibody can bind to an epitope within,or overlapping with, a fragment of a human complement component C5protein, the fragment containing, or consisting of, any one of thefollowing amino acid sequences (which are exemplary antigenic fragmentsof SEQ ID NO:1):

(SEQ ID NO: 7) NFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSD; (SEQ ID NO: 8)SESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVLPLEIGLHNINFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKEGMLSIMSYRNAD YSYS; (SEQ ID NO: 9)SHKDMQLGRLHMKTLLPVSKPEIRSYFPES; (SEQ ID NO: 10)SHKDMQLGRLHMKTLLPVSKPEIRSYFPESWLWEVHLVPRRKQLQFALPDSLTTWEIQGIGISNTGICVADTVKAKVFKDVFLEMNIPYSVVRGEQIQLKGTVYNYRTSGMQFCVKMSAVEGICTSESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVLPLEIGLHNINFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKEGMLSIMSYRNADYSYS; and (SEQ ID NO: 11) DHQGTKSSKCVRQKVEG.

Additional exemplary antigenic fragments of human complement componentC5 are disclosed in, e.g., U.S. Pat. No. 6,355,245, the disclosure ofwhich is incorporated herein by reference.

In some embodiments, the anti-C5 antibody specifically binds to a humancomplement component C5 protein (e.g., the human C5 protein having theamino acid sequence depicted in SEQ ID NO:1). The terms “specificbinding” or “specifically binds” refer to two molecules forming acomplex (e.g., a complex between an antibody and a complement componentC5 protein) that is relatively stable under physiologic conditions.Typically, binding is considered specific when the association constant(K_(a)) is higher than 10⁶ M⁻¹. Thus, an antibody can specifically bindto a C5 protein with a K_(a) of at least (or greater than) 10⁶ (e.g., atleast or greater than 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ 10¹², 10¹³, 10¹⁴, or10¹⁵ or higher) M⁻¹. Examples of antibodies that specifically bind to ahuman complement component C5 protein are described in, e.g., U.S. Pat.No. 6,355,245, the disclosure of which is incorporated herein byreference in its entirety.

Methods for determining whether an antibody binds to a protein antigenand/or the affinity for an antibody to a protein antigen are known inthe art. For example, the binding of an antibody to a protein antigencan be detected and/or quantified using a variety of techniques such as,but not limited to, Western blot, dot blot, plasmon surface resonancemethod (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Swedenand Piscataway, N.J.), or enzyme-linked immunosorbent assays (ELISA).See, e.g., Harlow and Lane (1988) “Antibodies: A Laboratory Manual” ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Benny K. C. Lo(2004) “Antibody Engineering: Methods and Protocols,” Humana Press(ISBN: 1588290921); Borrebaek (1992) “Antibody Engineering, A PracticalGuide,” W.H. Freeman and Co., NY; Borrebaek (1995) “AntibodyEngineering,” 2^(nd) Edition, Oxford University Press, NY, Oxford; Johneet al. (1993) J. Immunol. Meth. 160:191-198; Jonsson et al. (1993) Ann.Biol. Clin. 51:19-26; and Jonsson et al. (1991) Biotechniques11:620-627. See also, U.S. Pat. No. 6,355,245.

In some embodiments, the anti-C5 antibody can crossblock binding ofanother antibody that binds to an epitope within, or overlapping with, ahuman complement component C5 protein. In some embodiments, the anti-C5antibody can crossblock binding of an antibody that binds to an epitopewithin, or overlapping with, a peptide fragment of a human complementcomponent C5 protein. The peptide fragment can be a fragment of a humancomplement component C5 protein having the amino acid sequence depictedin any one of SEQ ID NOS:1-11. For example, the peptide fragment cancontain, or consist of, the following amino acid sequence:

(SEQ ID NO: 5) VIDHQGTKSSKCVRQKVEGSS.

As used herein, the term “crossblocking antibody” refers to an antibodythat lowers the amount of binding of anti-C5 antibody to an epitope on acomplement component C5 protein relative to the amount of binding of theanti-C5 antibody to the epitope in the absence of the antibody. Suitablemethods for determining whether a first antibody crossblocks binding ofa second antibody to an epitope are known in the art. For example,crossblocking antibodies can be identified by comparing the binding ofthe 5G1.1 anti-C5 monoclonal antibody (produced by the hybridoma cellline ATCC designation HB-11625; see U.S. Pat. No. 6,355,245) in thepresence and absence of a test antibody. Decreased binding of the 5G1.1antibody in the presence of the test antibody as compared to binding ofthe 5G1.1 antibody in the absence of the test antibody indicates thetest antibody is a crossblocking antibody.

Methods for identifying the epitope to which a particular antibody(e.g., an anti-C5 antibody) binds are also known in the art. Forexample, the binding epitope of an anti-C5 antibody can be identified bymeasuring the binding of the antibody to several (e.g., three, four,five, six, seven, eight, nine, 10, 15, 20, or 30 or more) overlappingpeptide fragments of a complement component C5 protein (e.g., severaloverlapping fragments of a protein having the amino acid sequencedepicted in any one of SEQ ID NOs:1-11). Each of the differentoverlapping peptides is then bound to a unique address on a solidsupport, e.g., separate wells of a multi-well assay plate. Next, theanti-C5 antibody is interrogated by contacting it to each of thepeptides in the assay plate for an amount of time and under conditionsthat allow for the antibody to bind to its epitope. Unbound anti-C5antibody is removed by washing each of the wells. Next, adetectably-labeled secondary antibody that binds to the anti-C5antibody, if present in a well of the plate, is contacted to each of thewells, and unbound secondary antibody is removed by washing steps. Thepresence or amount of the detectable signal produced by thedetectably-labeled secondary antibody in a well is an indication thatthe anti-C5 antibody binds to the particular peptide fragment associatedwith the well. See, e.g., Harlow and Lane (supra), Benny K. C. Lo(supra), and U.S. Patent Application Publication No. 20060153836, thedisclosure of which is incorporated by reference in its entirety. Aparticular epitope to which an antibody binds can also be identifiedusing BIAcore chromatographic techniques (see, e.g., PharmaciaBIAtechnology Handbook, “Epitope Mapping,” Section 6.3.2, (May 1994);and Johne et al. (1993) J. Immunol. Methods 160:20191-8).

The anti-C5 antibodies described herein can have activity in blockingthe generation or activity of the C5a and/or C5b active fragments of acomplement component C5 protein (e.g., a human C5 protein). Through thisblocking effect, the anti-C5 antibodies inhibit, e.g., theproinflammatory effects of C5a and the generation of the C5b-9 membraneattack complex (MAC) at the surface of a cell. Anti-C5 antibodies thathave the ability to block the generation of C5a are described in, e.g.,Moongkarndi et al. (1982) Immunobiol. 162:397 and Moongkarndi et al.(1983) Immunobiol. 165:323.

In some embodiments, an anti-C5 antibody, or antigen-binding fragmentthereof, can reduce the ability of a C5 protein to bind to humancomplement component C3b (e.g., C3b present in an AP or CP C5 convertasecomplex) by greater than 50 (e.g., greater than 55, 60, 65, 70, 75, 80,85, 90, or 95 or more) %. In some embodiments, upon binding to a C5protein, the anti-C5 antibody or antigen-binding fragment thereof canreduce the ability of the C5 protein to bind to complement component C4b(e.g., C4b present in a CP C5 convertase) by greater than 50 (e.g.,greater than 55, 60, 65, 70, 75, 80, 85, 90, or 95 or more) %. Methodsfor determining whether an antibody can block the generation or activityof the C5a and/or C5b active fragments of a complement component C5protein, or binding to complement component C4b or C3b, are known in theart and described in, e.g., U.S. Pat. No. 6,355,245 and Wurzner et al.(1991) Complement Inflamm 8:328-340. (See also below.)

In some embodiments, an anti-C5 antibody binds to an amino-terminalregion of the alpha chain of a complement component C5 protein, but doesnot bind to free C5a. Epitopes for an anti-C5 antibody within theamino-terminal region of the alpha chain include, e.g., epitopes withinthe human sequence

(SEQ ID NO: 5) VIDHQGTKSSKCVRQKVEGSS.

In some embodiments, the composition comprises, and/or the antibody is,eculizumab (Soliris®; Alexion Pharmaceuticals, Inc., Cheshire, Conn.).(See, e.g., Kaplan (2002) Curr Opin Investig Drugs 3(7):1017-23; Hill(2005) Clin Adv Hematol Oncol 3(11):849-50; and Rother et al. (2007)Nature Biotechnology 25(11):1256-1488.)

In some embodiments, the composition comprises, and/or the antibody is,pexelizumab (Alexion Pharmaceuticals, Inc., Cheshire, Conn.). (See,e.g., Whiss (2002) Curr Opin Investig Drugs 3(6):870-7; Patel et al.(2005) Drugs Today (Barc) 41(3):165-70; and Thomas et al. (1996) Mol.Immunol. 33(17-18):1389-401.)

In some embodiments, the C5 inhibitor is an antibody that binds to C5a(sometimes referred to herein as “an anti-C5a antibody”). In someembodiments, the antibody binds to C5a, but not to full-length C5. Asdiscussed above, the proform of C5, a 1676 amino acid residue precursorprotein, is processed by a series of proteolytic cleavage events. Thefirst 18 peptides (numbered −18 to −1) constitute a signal peptide thatis cleaved from the precursor protein. The remaining 1658 amino acidprotein is cleaved in two places to form the alpha and beta chains. Thefirst cleavage event occurs between amino acid residues 655 and 656. Thesecond cleavage occurs between amino acid residues 659 to 660. The twocleavage events result in the formation of three distinct polypeptidefragments: (i) a fragment comprising amino acids 1 to 655, which isreferred to as the beta chain; (ii) a fragment comprising amino acids660 to 1658, which is referred to as the alpha chain; and (iii) atetrapeptide fragment consisting of amino acids 656 to 659. The alphachain and the beta chain polypeptide fragments are connected to eachother via disulfide bond and constitute the mature C5 protein. The CP orAP C5 convertase activates mature C5 by cleaving the alpha chain betweenresidues 733 and 734, which results in the liberation of C5a fragment(amino acids 660 to 733). The remaining portion of mature C5 is fragmentC5b, which contains the residues 734 to 1658 of the alpha chaindisulfide bonded to the beta chain.

In vivo, C5a is rapidly metabolized by a serum enzyme, carboxypeptidaseB, to a 73 amino acid form termed “C5a des-Arg,” which has lost thecarboxyterminal arginine residue. Accordingly, in some embodiments, anantibody that binds to C5a also binds to desarginated C5a. In someembodiments, an antibody that binds to C5a does not bind to desarginatedC5a.

In some embodiments, the C5 inhibitor is an antibody that binds to aneoepitope present in C5a, i.e., an epitope that becomes exposed uponthe liberation of C5a from the alpha chain fragment of mature C5.Antibodies that bind to C5a (e.g., a neo-epitope present in C5a) areknown in the art as are methods for producing such antibodies. Forexample, an antibody that binds to C5a can have the binding specificityof a C5a neoepitope specific antibody described in any one of, e.g., PCTPublication No. WO 01/15731; Ames et al. (1994) J Immunol152(9):4572-4581; Inoue (1989) Complement Inflamm 6(3):219-222; and U.S.Pat. No. 6,866,845. In another example, an antibody that binds to C5acan have the binding specificity of a commercial C5a neoepitope-specificantibody such as, but not limited to, sc-52633 (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.), 152-1486 (BD Pharmingen/BDBiosciences), ab11877 (Abcam, Cambridge, Mass.), and HM2079 (clone 2952;HyCult Biotechnology, the Netherlands). In some embodiments, an antibodythat binds to C5a can crossblock the binding of any of theaforementioned C5a neoepitope-specific antibodies.

In some embodiments, the C5 inhibitor can be an antibody that binds to amammalian (e.g., human) C5a protein. For example, the antibody can bindto a human C5a protein having the following amino acid sequence:

(SEQ ID NO: 12) TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDMQLGR.The antibody can bind to human C5a at an epitope within or overlappingwith the amino acid sequence:

(SEQ ID NO: 13) CCYDGACVNNDETCEQRAAR; (SEQ ID NO: 14)KCCYDGACVNNDETCEQR; (SEQ ID NO: 15) VNNDETCEQR; (SEQ ID NO: 16) VNNDET;(SEQ ID NO: 17) AARISLGPR; (SEQ ID NO: 18) CCYDGACVNNDETCEQRAA;(SEQ ID NO: 19) CCYDGACVNNDETCEQRA; (SEQ ID NO: 20) CCYDGACVNNDETCEQR;(SEQ ID NO: 21) CCYDGACVNNDETCEQ; (SEQ ID NO: 22) CCYDGACVNNDETCE;(SEQ ID NO: 23) CYDGACVNNDETCEQRAAR; (SEQ ID NO: 24) YDGACVNNDETCEQRAAR;or  (SEQ ID NO: 25) CYDGACVNNDETCEQRAAR.In some embodiments, an antibody can bind to a human C5a protein orfragment thereof containing an amino acid sequence that contains, orconsists of, at least four (e.g., at least four, five, six, seven,eight, nine, 10, 11, 12, 13, 14, 15, 16, or 17 or more) consecutiveamino acids depicted in any one of SEQ ID NOs:12-25. Additional C5aprotein fragments to which an antibody described herein can bind andmethods for generating suitable C5a-specific antigen combining sites areset forth in, e.g., U.S. Pat. No. 4,686,100, the disclosure of which isincorporated herein by reference in its entirety.

In some embodiments, the binding of an antibody to C5a can inhibit thebiological activity of C5a. Methods for measuring C5a activity include,e.g., chemotaxis assays, RIAs, or ELISAs (see, e.g., Ward and Zvaifler(1971) J Clin Invest 50(3):606-16 and Wurzner et al. (1991) ComplementInflamm 8:328-340). In some embodiments, the binding of an antibody toC5a can inhibit the interaction between C5a and C5aR1. Suitable methodsfor detecting and/or measuring the interaction between C5a and C5aR1 (inthe presence and absence of an antibody) are known in the art anddescribed in, e.g., Mary and Boulay (1993) Eur J Haematol 51(5):282-287;Kaneko et al. (1995) Immunology 86(1):149-154; Giannini et al. (1995) JBiol Chem 270(32):19166-19172; and U.S. Patent Application PublicationNo. 20060160726. For example, the binding of detectably labeled (e.g.,radioactively labeled) C5a to C5aR1-expressing peripheral bloodmononuclear cells can be evaluated in the presence and absence of anantibody. A decrease in the amount of detectably-labeled C5a that bindsto C5aR1 in the presence of the antibody, as compared to the amount ofbinding in the absence of the antibody, is an indication that theantibody inhibits the interaction between C5a and C5aR1. In someembodiments, the binding of an antibody to C5a can inhibit theinteraction between C5a and C5L2 (see below). Methods for detectingand/or measuring the interaction between C5a and C5L2 are known in theart and described in, e.g., Ward (2009) J Mol Med 87(4):375-378 and Chenet al. (2007) Nature 446(7132):203-207 (see below).

In some embodiments, the C5 inhibitor is an antibody that binds to C5b(sometimes referred to herein as “an anti-C5b antibody”). In someembodiments, the antibody binds to C5b, but does not bind to full-lengthC5. The structure of C5b is described above and also detailed in, e.g.,Müller-Eberhard (1985) Biochem Soc Symp 50:235-246; Yamamoto and Gewurz(1978) J Immunol 120(6):2008-2015; and Haviland et al. (1991), supra. Asdescribed above, C5b combines with C6, C7, and C8 to form the C5b-8complex at the surface of the target cell. Protein complex intermediatesformed during the series of combinations include C5b-6 (including C5band C6), C5b-7 (including C5b, C6, and C7), and C5b-8 (including C5b,C6, C7, and C8). Upon binding of several C9 molecules, the membraneattack complex (MAC, C5b-9 terminal complement complex (TCC)) is formed.When sufficient numbers of MACs insert into target cell membranes, theopenings they create (MAC pores) mediate rapid osmotic lysis of thetarget cells.

In some embodiments, the binding of an antibody to C5b can inhibit theinteraction between C5b and C6. In some embodiments, the binding of theantibody to C5b can inhibit the assembly or activity of the C5b-9MAC-TCC. In some embodiments, the binding of an antibody to C5b caninhibit complement-dependent cell lysis (e.g., in vitro and/or in vivo).Suitable methods for evaluating whether an antibody inhibitscomplement-dependent lysis include, e.g., hemolytic assays or otherfunctional assays for detecting the activity of soluble C5b-9. Forexample, a reduction in the cell-lysing ability of complement in thepresence of an antibody can be measured by a hemolysis assay describedby Kabat and Mayer (eds.), “Experimental Immunochemistry, 2^(nd)Edition,” 135-240, Springfield, Ill., CC Thomas (1961), pages 135-139,or a conventional variation of that assay such as the chickenerythrocyte hemolysis method as described in, e.g., Hillmen et al.(2004) N Engl J Med 350(6):552.

Antibodies that bind to C5b as well as methods for making suchantibodies are known in the art. See, e.g., U.S. Pat. No. 6,355,245.Commercially available anti-C5b antibodies are available from a numberof vendors including, e.g., Hycult Biotechnology (catalogue number:HM2080; clone 568) and Abcam™ (ab46151 or ab46168).

In some embodiments, the C5 inhibitor is an antibody that binds to amammalian (e.g., human) form of C5b. For example, the antibody can bindto a portion of a human C5b protein having the following amino acidsequence:

(SEQ ID NO: 4) QEQTYVISAPKIFRVGASENIVIQVYGYTEAFDATISIKSYPDKKFSYSSGHVHLSSENKFQNSAILTIQPKQLPGGQNPVSYVYLEVVSKHFSKSKRMPITYDNGFLFIHTDKPVYTPDQSVKVRVYSLNDDLKPAKRETVLTFIDPEGSEVDMVEEIDHIGIISFPDFKIPSNPRYGMWTIKAKYKEDFSTTGTAYFEVKEYVLPHFSVSIEPEYNFIGYKNFKNFEITIKARYFYNKVVTEADVYITFGIREDLKDDQKEMMQTAMQNTMLINGIAQVTFDSETAVKELSYYSLEDLNNKYLYIAVTVIESTGGFSEEAEIPGIKYVLSPYKLNLVATPLFLKPGIPYPIKVQVKDSLDQLVGGVPVILNAQTIDVNQETSDLDPSKSVTRVDDGVASFVLNLPSGVTVLEFNVKTDAPDLPEENQAREGYRAIAYSSLSQSYLYIDWTDNHKALLVGEHLNIIVTPKSPYIDKITHYNYLILSKGKIIHFGTREKFSDASYQSINIPVTQNMVPSSRLLVYYIVTGEQTAELVSDSVWLNIEEKCGNQLQVHLSPDADAYSPGQTVSLNMATGMDSWVALAAVDSAVYGVQRGAKKPLERVFQFLEKSDLGCGAGGGLNNANVFHLAGLTFLT NANADDSQENDEPCKEIL.In some embodiments, the antibody can bind to a portion of a human C5bprotein having the following amino acid sequence:

(SEQ ID NO: 26) LHMKTLLPVSKPEIRSYFPESWLWEVHLVPRRKQLQFALPDSLTTWEIQGIGISNTGICVADTVKAKVFKDVFLEMNIPYSVVRGEQIQLKGTVYNYRTSGMQFCVKMSAVEGICTSESPVIDHQGTKSSKCVRQKVEGSSSHLVTFTVLPLEIGLHNINFSLETWFGKEILVKTLRVVPEGVKRESYSGVTLDPRGIYGTISRRKEFPYRIPLDLVPKTEIKRILSVKGLLVGEILSAVLSQEGINILTHLPKGSAEAELMSVVPVFYVFHYLETGNHWNIFHSDPLIEKQKLKKKLKEGMLSIMSYRNADYSYSVWKGGSASTWLTAFALRVLGQVNKYVEQNQNSICNSLLWLVENYQLDNGSFKENSQYQPIKLQGTLPVEARENSLYLTAFTVIGIRKAFDICPLVKIDTALIKADNFLLENTLPAQSTFTLAISAYALSLGDKTHPQFRSIVSALKREALVKGNPPIYRFWKDNLQHKDSSVPNTGTARMVETTAYALLTSLNLKDINYVNPVIKWLSEEQRYGGGFYSTQDTINAIEGLTEYSLLVKQLRLSMDIDVSYKHKGALHNYKMTDKNFLGRPVEVLLNDDLIVSTGFGSGLATVHVTTVVHKTSTSEEVCSFYLKIDTQDIEASHYRGYGNSDYKRIVACASYKPSREESSSGSSHAVMDISLPTGISANEEDLKALVEGVDQLFTDYQIKDGHVILQLNSIPSSDFLCVRFRIFELFEVGFLSPATFTVYEYHRPDKQCTMFYSTSNIKIQKVCEGAACKCVEADCGQMQEELDLTISAETRKQTACKPEIAYAYKVSITSITVENVFVKYKATLLDIYKTGEAVAEKDSEITFIKKVTCTNAELVKGRQYLIMGKEALQIKYNFSFRYIYPLDSLTWIEYWPRDTTCSSCQAFLANLDEFAEDIFLNGC.In some embodiments, the antibody can bind to human C5b protein orfragment thereof containing an amino acid sequence that contains, orconsists of, at least four (e.g., at least four, five, six, seven,eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more)consecutive amino acids depicted in SEQ ID NO:4 or SEQ ID NO:26.

Additional exemplary sub-fragments of human C5b or C5a to which a C5inhibitor antibody can bind are disclosed in, e.g., U.S. Pat. No.6,355,245, the disclosure of which is incorporated herein by reference.

In some embodiments, the inhibitor is an antibody that specificallybinds to a C5a polypeptide (e.g., the human C5a polypeptide having theamino acid sequence depicted in SEQ ID NO:12). In some embodiments, theinhibitor is an antibody that specifically binds to a C5b polypeptide.

Methods for determining whether a particular agent is an inhibitor ofhuman complement component C5 are described herein and are known in theart. For example, the concentration and/or physiologic activity of C5aand C5b in a body fluid can be measured by methods well known in theart. Methods for measuring C5a concentration or activity include, e.g.,chemotaxis assays, RIAs, or ELISAs (see, e.g., Ward and Zvaifler (1971)J Clin Invest. 50(3):606-16 and Wurzner et al. (1991) ComplementInflamm. 8:328-340). For C5b, hemolytic assays or assays for solubleC5b-9 as discussed herein can be used. Other assays known in the art canalso be used. Using assays of these or other suitable types, candidateagents capable of inhibiting human complement component C5 such as ananti-C5 antibody, can be screened in order to, e.g., identify compoundsthat are useful in the methods described herein and determine theappropriate dosage levels of such compounds.

Methods for detecting inhibition of expression of mRNA or protein (e.g.,inhibition of human C5 protein expression or expression of an mRNAencoding human C5 protein) are well known in the art of molecularbiology and include, e.g., Northern blot and RT-PCR (or quantitativeRT-PCR) techniques for mRNA and for protein detection, Western blot, dotblot, or ELISA techniques. (See, e.g., Sambrook et al. (1989) “MolecularCloning: A Laboratory Manual, 2^(nd) Edition,” Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.)

Methods for determining whether a candidate compound inhibits thecleavage of human C5 into forms C5a and C5b are known in the art anddescribed in, e.g., Moongkarndi et al. (1982) Immunobiol. 162:397;Moongkarndi et al. (1983) Immunobiol. 165:323; Isenman et al. (1980) J.Immunol. 124(1):326-31; Thomas et al. (1996) Mol. Immunol.33(17-18):1389-401; and Evans et al. (1995) Mol. Immunol.32(16):1183-95.

Inhibition of human complement component C5 can also reduce thecell-lysing ability of complement in a subject's body fluids. Suchreductions of the cell-lysing ability of complement present can bemeasured by methods well known in the art such as, for example, by aconventional hemolytic assay such as the hemolysis assay described byKabat and Mayer (eds), “Experimental Immunochemistry, 2^(nd) Edition,”135-240, Springfield, Ill., CC Thomas (1961), pages 135-139, or aconventional variation of that assay such as the chicken erythrocytehemolysis method as described in, e.g., Hillmen et al. (2004) N Engl JMed 350(6):552.

Pharmaceutical Compositions and Formulations.

The compositions containing a complement inhibitor (e.g., an inhibitorof human complement component C5 such as an anti-C5 antibody orantigen-binding fragment thereof) can be formulated as a pharmaceuticalcomposition, e.g., for administration to a subject to treat aHUS, CAPS,Degos disease, or TMA. The pharmaceutical compositions will generallyinclude a pharmaceutically acceptable carrier. As used herein, a“pharmaceutically acceptable carrier” refers to, and includes, any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The compositions can include apharmaceutically acceptable salt, e.g., an acid addition salt or a baseaddition salt (see e.g., Berge et al. (1977) J. Pharm. Sci. 66:1-19).

The compositions can be formulated according to standard methods.Pharmaceutical formulation is a well-established art, and is furtherdescribed in, e.g., Gennaro (2000) “Remington: The Science and Practiceof Pharmacy,” 20^(th) Edition, Lippincott, Williams & Wilkins (ISBN:0683306472); Ansel et al. (1999) “Pharmaceutical Dosage Forms and DrugDelivery Systems,” 7^(th) Edition, Lippincott Williams & WilkinsPublishers (ISBN: 0683305727); and Kibbe (2000) “Handbook ofPharmaceutical Excipients American Pharmaceutical Association,” 3^(rd)Edition (ISBN: 091733096X). In some embodiments, a composition can beformulated, for example, as a buffered solution at a suitableconcentration and suitable for storage at 2-8° C. In some embodiments, acomposition can be formulated for storage at a temperature below 0° C.(e.g., −20° C. or −80° C.).

The pharmaceutical compositions can be in a variety of forms. Theseforms include, e.g., liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends, in part, on the intended mode ofadministration and therapeutic application. For example, compositionscontaining an anti-C5 antibody intended for systemic or local deliverycan be in the form of injectable or infusible solutions. Accordingly,the compositions can be formulated for administration by a parenteralmode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscularinjection). “Parenteral administration,” “administered parenterally,”and other grammatically equivalent phrases, as used herein, refer tomodes of administration other than enteral and topical administration,usually by injection, and include, without limitation, intravenous,intranasal, intraocular, pulmonary, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intrapulmonary, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural, intracerebral, intracranial, intracarotid and intrasternalinjection and infusion (see below).

The compositions can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an antibody described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating aninhibitor of human complement component C5 (e.g., an anti-C5 antibody)described herein into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods for preparation include vacuum drying andfreeze-drying that yield a powder of the antibody described herein plusany additional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition areagent that delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the C5 inhibitor (e.g., an anti-C5 antibody orantigen-binding fragment thereof) can be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are known in the art. (See, e.g., J. R. Robinson(1978) “Sustained and Controlled Release Drug Delivery Systems,” MarcelDekker, Inc., New York.)

In some embodiments, an antibody described herein can be formulated in acomposition suitable for intrapulmonary administration (e.g., foradministration via nebulizer) to a mammal such as a human. Methods forpreparing such compositions are well known in the art and described in,e.g., U.S. Patent Application Publication No. 20080202513; U.S. Pat.Nos. 7,112,341 and 6,019,968; and PCT Publication Nos. WO 00/061178 andWO 06/122257, the disclosures of each of which are incorporated hereinby reference in their entirety. Dry powder inhaler formulations andsuitable systems for administration of the formulations are describedin, e.g., U.S. Patent Application Publication No. 20070235029, PCTPublication No. WO 00/69887; and U.S. Pat. No. 5,997,848.

In some embodiments, an inhibitor of human C5 (e.g., an anti-C5 antibodyor antigen-binding fragment thereof) described herein can be modified,e.g., with a moiety that improves its stabilization and/or retention incirculation, e.g., in blood, serum, or other tissues. The stabilizationmoiety can improve the stability, or retention of, the antibody by atleast 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more)fold.

The nucleic acid inhibitors of human complement component C5 describedherein (e.g., an anti-sense nucleic acid or siRNA) can be incorporatedinto a gene construct to be used as a part of a gene therapy protocol todeliver nucleic acids that can be used to express and produce agentswithin cells. Expression constructs of such components may beadministered in any biologically effective carrier, e.g. any formulationor composition capable of effectively delivering the component gene tocells in vivo. Approaches include insertion of the subject gene in viralvectors including recombinant retroviruses, adenovirus, adeno-associatedvirus, lentivirus, and herpes simplex virus-1 (HSV-1), or recombinantbacterial or eukaryotic plasmids. Viral vectors can transfect cellsdirectly; plasmid DNA can be delivered with the help of, for example,cationic liposomes (lipofectin) or derivatized (e.g., antibodyconjugated), polylysine conjugates, gramicidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo. (See also, “Ex vivo Approaches,” below.) Examples of suitableretroviruses include pLJ, pZIP, pWE and pEM which are known to thoseskilled in the art (see, e.g., Eglitis et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT PublicationNos. WO89/07136, WO89/02468, WO89/05345, and WO92/07573). Another viralgene delivery system utilizes adenovirus-derived vectors (see, e.g.,Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991)Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155).Suitable adenoviral vectors derived from the adenovirus strain Ad type 5dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) areknown to those skilled in the art. Yet another viral vector systemuseful for delivery of the subject gene is the adeno-associated virus(AAV). See, e.g., Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol.7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlinet al. (1989) J. Virol 62: 1963-1973.

In some embodiments, more than one (e.g., two, three, four, five, six,seven, eight, nine, or 10 or more) inhibitor(s) (e.g., one or moreinhibitors of human C5) can be co-formulated. For example, a C5-specificsiRNA and an anti-C5 antibody can be formulated together.

In some embodiments, an inhibitor of human complement (e.g., aninhibitor human C5 such as an anti-C5 antibody or antigen-bindingfragment thereof) described herein can be formulated with one or moreadditional active agents useful for treating a complement-associateddisorder (e.g., any of the complement-associated disorders describedherein such as APS, CAPS, aHUS, Degos disease, HELLP syndrome) orameliorating a symptom thereof. For example, an anti-C5 antibody can beformulated with an antihypertensive, an anticoagulant, and/or a steroid(e.g., a corticosteroid). Examples of anticoagulants include, e.g.,warfarin (Coumadin), aspirin, heparin, phenindione, fondaparinux,idraparinux, and thrombin inhibitors (e.g., argatroban, lepirudin,bivalirudin, or dabigatran). An inhibitor of human C5 (e.g., an anti-C5antibody, an anti-C5a antibody, or an anti-C5b antibody) can also beformulated with a fibrinolytic agent (e.g., ancrod, E-aminocaproic acid,antiplasmin-a₁, prostacyclin, and defibrotide), cyclophosphamide, or ananti-cytokine agent for the treatment of CAPS. Anti-cytokine agentsinclude, e.g., antibodies or soluble receptors that bind to and modulatethe activity of cytokine (e.g., a pro-inflammatory cytokine such asTNF). Examples of anti-cytokine agents include, e.g., a TNF inhibitorsuch as a soluble TNF receptor (e.g., etanercept; Enbrel®) or ananti-TNF antibody (e.g., infliximab; Remicade®). In some embodiments,the inhibitor can be formulated with, or for use with, an anti-CD20agent such as rituximab (Rituxan™; Biogen Idec, Cambridge, Mass.). Insome embodiments, the inhibitor of human C5 can be formulated foradministration to a subject along with intravenous immunoglobulintherapy (IVIG) or with plasma exchange.

When the inhibitor of human C5 is to be used in combination with asecond active agent, or when two or more inhibitors of human C5 are tobe used (e.g., an anti-C5a antibody and an anti-C5b antibody), theagents can be formulated separately or together. For example, therespective pharmaceutical compositions can be mixed, e.g., just prior toadministration, and administered together or can be administeredseparately, e.g., at the same or different times (see below).

As described above, a composition can be formulated such that itincludes a therapeutically effective amount of an inhibitor of human C5(e.g., an anti-C5 antibody or antigen-binding fragment thereof) or thecomposition can be formulated to include a sub-therapeutic amount of theinhibitor and a sub-therapeutic amount of one or more additional activeagents such that the components in total are therapeutically effectivefor treating a complement-associated disorder such as any of thosedescribed herein. In some embodiments, a composition can be formulatedto include two or more inhibitors of human C5, each at sub-therapeuticdoses, such that the inhibitors in total are at a concentration that istherapeutically effective for treating a complement-associated disordersuch as, e.g., aHUS, CAPS, Degos disease, or HELLP syndrome. Methods fordetermining a therapeutically effective dose (e.g., a therapeuticallyeffective dose of an anti-C5 antibody) are known in the art anddescribed herein.

Methods for Producing an Antibody

Suitable methods for producing an antibody (e.g., an anti-C5 antibody,an anti-C5a antibody, and/or an anti-C5b antibody), or antigen-bindingfragments thereof, in accordance with the disclosure are known in theart (see, e.g., U.S. Pat. No. 6,355,245) and described herein. Forexample, monoclonal anti-C5 antibodies may be generated using complementcomponent C5-expressing cells, a C5 polypeptide, or an antigenicfragment of C5 polypeptide (e.g., C5a or C5b), as an immunogen, thusraising an immune response in animals from which antibody-producingcells and in turn monoclonal antibodies may be isolated. The sequence ofsuch antibodies may be determined and the antibodies or variants thereofproduced by recombinant techniques. Recombinant techniques may be usedto produce chimeric, CDR-grafted, humanized and fully human antibodiesbased on the sequence of the monoclonal antibodies as well aspolypeptides capable of binding to human complement component C5.

Moreover, antibodies derived from recombinant libraries (“phageantibodies”) may be selected using, e.g., C5-expressing cells, orpolypeptides derived therefrom, as bait to isolate the antibodies orpolypeptides on the basis of target specificity. The production andisolation of non-human and chimeric anti-C5 antibodies are well withinthe purview of the skilled artisan.

Recombinant DNA technology can be used to modify one or morecharacteristics of the antibodies produced in non-human cells. Thus,chimeric antibodies can be constructed in order to decrease theimmunogenicity thereof in diagnostic or therapeutic applications.Moreover, immunogenicity can be minimized by humanizing the antibodiesby CDR grafting and, optionally, framework modification. See, U.S. Pat.Nos. 5,225,539 and 7,393,648, the contents of each of which areincorporated herein by reference.

Antibodies can be obtained from animal serum or, in the case ofmonoclonal antibodies or fragments thereof, produced in cell culture.Recombinant DNA technology can be used to produce the antibodiesaccording to established procedure, including procedures in bacterial orpreferably mammalian cell culture. The selected cell culture systempreferably secretes the antibody product.

In another embodiment, a process for the production of an antibodydisclosed herein includes culturing a host, e.g., E. coli or a mammaliancell, which has been transformed with a hybrid vector. The vectorincludes one or more expression cassettes containing a promoter operablylinked to a first DNA sequence encoding a signal peptide linked in theproper reading frame to a second DNA sequence encoding the antibodyprotein. The antibody protein is then collected and isolated.Optionally, the expression cassette may include a promoter operablylinked to polycistronic (e.g., bicistronic) DNA sequences encodingantibody proteins each individually operably linked to a signal peptidein the proper reading frame.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which include the customarystandard culture media (such as, for example Dulbecco's Modified EagleMedium (DMEM) or RPMI 1640 medium), optionally replenished by amammalian serum (e.g. fetal calf serum), or trace elements and growthsustaining supplements (e.g. feeder cells such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages,2-aminoethanol, insulin, transferrin, low density lipoprotein, oleicacid, or the like). Multiplication of host cells which are bacterialcells or yeast cells is likewise carried out in suitable culture mediaknown in the art. For example, for bacteria suitable culture mediainclude medium LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, orM9 Minimal Medium. For yeast, suitable culture media include medium YPD,YEPD, Minimal Medium, or Complete Minimal propout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up production to give large amounts of the desiredantibodies. Techniques for bacterial cell, yeast, plant, or mammaliancell cultivation are known in the art and include homogeneous suspensionculture (e.g. in an airlift reactor or in a continuous stirrer reactor),and immobilized or entrapped cell culture (e.g. in hollow fibers,microcapsules, on agarose microbeads or ceramic cartridges).

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumors. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristane. After one to two weeks, asciticfluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, the disclosures of which are all incorporated herein byreference. Techniques for the preparation of recombinant antibodymolecules are described in the above references and also in, e.g.:WO97/08320; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,508,717; Smith(1985) Science 225:1315-1317; Parmley and Smith (1988) Gene 73:305-318;De La Cruz et al. (1988) Journal of Biological Chemistry 263:4318-4322;U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,223,409; WO88/06630;WO92/15679; U.S. Pat. No. 5,780,279; U.S. Pat. No. 5,571,698; U.S. Pat.No. 6,040,136; Davis et al. (1999) Cancer Metastasis Rev. 18(4):421-5;and Taylor et al. (1992) Nucleic Acids Research 20: 6287-6295; Tomizukaet al. (2000) Proc. Natl. Acad. Sci. USA 97(2): 722-727, the contents ofeach of which are incorporated herein by reference in their entirety.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of complement componentC5-expressing cells, by immunoblotting, by an enzyme immunoassay, e.g. asandwich assay or a dot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid may be concentrated, e.g., byprecipitation with ammonium sulfate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with one or more surface polypeptides derived from acomplement component C5-expressing cell line, or with Protein-A or -G.

Another embodiment provides a process for the preparation of a bacterialcell line secreting antibodies directed against a C5 protein in asuitable mammal. For example a rabbit is immunized with pooled samplesfrom C5-expressing tissue or cells or C5 polypeptide or fragmentsthereof. A phage display library produced from the immunized rabbit isconstructed and panned for the desired antibodies in accordance withmethods well known in the art (such as, e.g., the methods disclosed inthe various references incorporated herein by reference).

Hybridoma cells secreting the monoclonal antibodies are also disclosed.The preferred hybridoma cells are genetically stable, secrete monoclonalantibodies described herein of the desired specificity, and can beexpanded from deep-frozen cultures by thawing and propagation in vitroor as ascites in vivo.

In another embodiment, a process is provided for the preparation of ahybridoma cell line secreting monoclonal antibodies against a complementcomponent C5 protein. In that process, a suitable mammal, for example aBalb/c mouse, is immunized with one or more polypeptides or antigenicfragments of C5 or with one or more polypeptides or antigenic fragmentsderived from a C5-expressing cell, the C5-expressing cell itself, or anantigenic carrier containing a purified polypeptide as described.Antibody-producing cells of the immunized mammal are grown briefly inculture or fused with cells of a suitable myeloma cell line. The hybridcells obtained in the fusion are cloned, and cell clones secreting thedesired antibodies are selected. For example, spleen cells of Balb/cmice immunized with a C5-expressing Chronic Lymphocytic Leukemia (CLL)cell line are fused with cells of the myeloma cell line PAI or themyeloma cell line Sp2/0-Ag 14. The obtained hybrid cells are thenscreened for secretion of the desired antibodies and positive hybridomacells are cloned.

Methods for preparing a hybridoma cell line include immunizing Balb/cmice by injecting subcutaneously and/or intraperitoneally an immunogeniccomposition containing human C5 protein (or an immunogenic fragmentthereof) several times, e.g., four to six times, over several months,e.g., between two and four months. Spleen cells from the immunized miceare taken two to four days after the last injection and fused with cellsof the myeloma cell line PAI in the presence of a fusion promoter,preferably polyethylene glycol. Preferably, the myeloma cells are fusedwith a three- to twenty-fold excess of spleen cells from the immunizedmice in a solution containing about 30% to about 50% polyethylene glycolof a molecular weight around 4000. After the fusion, the cells areexpanded in suitable culture media as described supra, supplemented witha selection medium, for example HAT medium, at regular intervals inorder to prevent normal myeloma cells from overgrowing the desiredhybridoma cells.

The antibodies and fragments thereof can be “chimeric.” Chimericantibodies and antigen-binding fragments thereof comprise portions fromtwo or more different species (e.g., mouse and human). Chimericantibodies can be produced with mouse variable regions of desiredspecificity spliced onto human constant domain gene segments (forexample, U.S. Pat. No. 4,816,567). In this manner, non-human antibodiescan be modified to make them more suitable for human clinicalapplication (e.g., methods for treating or preventing a complementassociated disorder in a human subject).

The monoclonal antibodies of the present disclosure include “humanized”forms of the non-human (e.g., mouse) antibodies. Humanized orCDR-grafted mAbs are particularly useful as therapeutic agents forhumans because they are not cleared from the circulation as rapidly asmouse antibodies and do not typically provoke an adverse immunereaction. Methods of preparing humanized antibodies are generally wellknown in the art. For example, humanization can be essentially performedfollowing the method of Winter and co-workers (see, e.g., Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;and Verhoeyen et al. (1988) Science 239:1534-1536), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Also see, e.g., Staelens et al. (2006) Mol Immunol43:1243-1257. In some embodiments, humanized forms of non-human (e.g.,mouse) antibodies are human antibodies (recipient antibody) in whichhypervariable (CDR) region residues of the recipient antibody arereplaced by hypervariable region residues from a non-human species(donor antibody) such as a mouse, rat, rabbit, or non-human primatehaving the desired specificity, affinity, and binding capacity. In someinstances, framework region residues of the human immunoglobulin arealso replaced by corresponding non-human residues (so called “backmutations”). In addition, phage display libraries can be used to varyamino acids at chosen positions within the antibody sequence. Theproperties of a humanized antibody are also affected by the choice ofthe human framework. Furthermore, humanized and chimerized antibodiescan be modified to comprise residues that are not found in the recipientantibody or in the donor antibody in order to further improve antibodyproperties, such as, for example, affinity or effector function.

Fully human antibodies are also provided in the disclosure. The term“human antibody” includes antibodies having variable and constantregions (if present) derived from human germline immunoglobulinsequences. Human antibodies can include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). However, the term “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences (i.e., humanized antibodies). Fully human or human antibodiesmay be derived from transgenic mice carrying human antibody genes(carrying the variable (V), diversity (D), joining (J), and constant (C)exons) or from human cells. For example, it is now possible to producetransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. (See, e.g., Jakobovits et al.(1993) Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al. (1993)Nature 362:255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; andDuchosal et al. (1992) Nature 355:258.) Transgenic mice strains can beengineered to contain gene sequences from unrearranged humanimmunoglobulin genes. The human sequences may code for both the heavyand light chains of human antibodies and would function correctly in themice, undergoing rearrangement to provide a wide antibody repertoiresimilar to that in humans. The transgenic mice can be immunized with thetarget protein (e.g., a complement component C5 protein, fragmentsthereof, or cells expressing C5 protein) to create a diverse array ofspecific antibodies and their encoding RNA. Nucleic acids encoding theantibody chain components of such antibodies may then be cloned from theanimal into a display vector. Typically, separate populations of nucleicacids encoding heavy and light chain sequences are cloned, and theseparate populations then recombined on insertion into the vector, suchthat any given copy of the vector receives a random combination of aheavy and a light chain. The vector is designed to express antibodychains so that they can be assembled and displayed on the outer surfaceof a display package containing the vector. For example, antibody chainscan be expressed as fusion proteins with a phage coat protein from theouter surface of the phage. Thereafter, display packages can be screenedfor display of antibodies binding to a target.

In addition, human antibodies can be derived from phage-displaylibraries (Hoogenboom et al. (1991) J. Mol. Biol. 227:381; Marks et al.(1991) J. Mol. Biol., 222:581-597; and Vaughan et al. (1996) NatureBiotech 14:309 (1996)). Synthetic phage libraries can be created whichuse randomized combinations of synthetic human antibody V-regions. Byselection on antigen fully human antibodies can be made in which theV-regions are very human-like in nature. See, e.g., U.S. Pat. Nos.6,794,132, 6,680,209, 4,634,666, and Ostberg et al. (1983), Hybridoma2:361-367, the contents of each of which are incorporated herein byreference in their entirety.

For the generation of human antibodies, also see Mendez et al. (1998)Nature Genetics 15:146-156, Green and Jakobovits (1998) J. Exp. Med.188:483-495, the disclosures of which are hereby incorporated byreference in their entirety. Human antibodies are further discussed anddelineated in U.S. Pat. Nos. 5,939,598; 6,673,986; 6,114,598; 6,075,181;6,162,963; 6,150,584; 6,713,610; and 6,657,103 as well as U.S. PatentApplication Publication Nos. 2003-0229905 A1, 2004-0010810 A1, US2004-0093622 A1, 2006-0040363 A1, 2005-0054055 A1, 2005-0076395 A1,2005-0287630 A1. See also International Publication Nos. WO 94/02602, WO96/34096, and WO 98/24893, and European Patent No. EP 0 463 151 B1. Thedisclosures of each of the above-cited patents, applications, andreferences are hereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825;5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318;5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825;5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, thedisclosures of which are hereby incorporated by reference. See alsoEuropean Patent No. 0 546 073 B1, International Patent Publication Nos.WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, thedisclosures of each of which are hereby incorporated by reference intheir entirety. See further Taylor et al. (1992) Nucleic Acids Res. 20:6287; Chen et al. (1993) Int. Immunol. 5: 647; Tuaillon et al. (1993)Proc. Natl. Acad. Sci. USA 90: 3720-4; Choi et al. (1993) NatureGenetics 4: 117; Lonberg et al. (1994) Nature 368: 856-859; Taylor etal. (1994) International Immunology 6: 579-591; Tuaillon et al. (1995)J. Immunol. 154: 6453-65; Fishwild et al. (1996) Nature Biotechnology14: 845; and Tuaillon et al. (2000) Eur. J. Immunol. 10: 2998-3005, thedisclosures of each of which are hereby incorporated by reference intheir entirety.

In certain embodiments, de-immunized anti-C5 antibodies orantigen-binding fragments thereof are provided. De-immunized antibodiesor antigen-binding fragments thereof are antibodies that have beenmodified so as to render the antibody or antigen-binding fragmentthereof non-immunogenic, or less immunogenic, to a given species (e.g.,to a human). De-immunization can be achieved by modifying the antibodyor antigen-binding fragment thereof utilizing any of a variety oftechniques known to those skilled in the art (see, e.g., PCT PublicationNos. WO 04/108158 and WO 00/34317). For example, an antibody orantigen-binding fragment thereof may be de-immunized by identifyingpotential T cell epitopes and/or B cell epitopes within the amino acidsequence of the antibody or antigen-binding fragment thereof andremoving one or more of the potential T cell epitopes and/or B cellepitopes from the antibody or antigen-binding fragment thereof, forexample, using recombinant techniques. The modified antibody orantigen-binding fragment thereof may then optionally be produced andtested to identify antibodies or antigen-binding fragments thereof thathave retained one or more desired biological activities, such as, forexample, binding affinity, but have reduced immunogenicity. Methods foridentifying potential T cell epitopes and/or B cell epitopes may becarried out using techniques known in the art, such as, for example,computational methods (see e.g., PCT Publication No. WO 02/069232), invitro or in silico techniques, and biological assays or physical methods(such as, for example, determination of the binding of peptides to MHCmolecules, determination of the binding of peptide:MHC complexes to theT cell receptors from the species to receive the antibody orantigen-binding fragment thereof, testing of the protein or peptideparts thereof using transgenic animals with the MHC molecules of thespecies to receive the antibody or antigen-binding fragment thereof, ortesting with transgenic animals reconstituted with immune system cellsfrom the species to receive the antibody or antigen-binding fragmentthereof, etc.). In various embodiments, the de-immunized anti-C5antibodies described herein include de-immunized antigen-bindingfragments, Fab, Fv, scFv, Fab′ and F(ab′)₂, monoclonal antibodies,murine antibodies, engineered antibodies (such as, for example,chimeric, single chain, CDR-grafted, humanized, fully human antibodies,and artificially selected antibodies), synthetic antibodies andsemi-synthetic antibodies.

In some embodiments, a recombinant DNA comprising an insert coding for aheavy chain variable domain and/or for a light chain variable domain ofan anti-C5 antibody or a C5 protein-expressing cell line is produced.The term DNA includes coding single stranded DNAs, double stranded DNAsconsisting of said coding DNAs and of complementary DNAs thereto, orthese complementary (single stranded) DNAs themselves.

Furthermore, a DNA encoding a heavy chain variable domain and/or a lightchain variable domain of anti-C5 antibodies can be enzymatically orchemically synthesized to contain the authentic DNA sequence coding fora heavy chain variable domain and/or for the light chain variabledomain, or a mutant thereof. A mutant of the authentic DNA is a DNAencoding a heavy chain variable domain and/or a light chain variabledomain of the above-mentioned antibodies in which one or more aminoacids are deleted, inserted, or exchanged with one or more other aminoacids. Preferably said modification(s) are outside the CDRs of the heavychain variable domain and/or of the light chain variable domain of theantibody in humanization and expression optimization applications. Theterm mutant DNA also embraces silent mutants wherein one or morenucleotides are replaced by other nucleotides with the new codons codingfor the same amino acid(s). The term mutant sequence also includes adegenerate sequence. Degenerate sequences are degenerate within themeaning of the genetic code in that an unlimited number of nucleotidesare replaced by other nucleotides without resulting in a change of theamino acid sequence originally encoded. Such degenerate sequences may beuseful due to their different restriction sites and/or frequency ofparticular codons which are preferred by the specific host, particularlyE. coli, to obtain an optimal expression of the heavy chain murinevariable domain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Recombinant DNAs including an insert coding for a heavy chain murinevariable domain of an anti-C5 antibody or a C5-expressing cell linefused to a human constant domain IgG, for example γ1, γ2, γ3 or γ4, inparticular embodiments γ1 or γ4, may be used. Recombinant DNAs includingan insert coding for a light chain murine variable domain of an antibodyfused to a human constant domain κ or λ, preferably κ, are alsoprovided.

Another embodiment pertains to recombinant DNAs coding for a recombinantpolypeptide wherein the heavy chain variable domain and the light chainvariable domain are linked by way of a spacer group, optionallycomprising a signal sequence facilitating the processing of the antibodyin the host cell and/or a DNA sequence encoding a peptide facilitatingthe purification of the antibody and/or a cleavage site and/or a peptidespacer and/or an agent.

Accordingly, the monoclonal antibodies or antigen-binding fragments ofthe disclosure can be naked antibodies or antigen-binding fragments thatare not conjugated to other agents, for example, a therapeutic agent ordetectable label. Alternatively, the monoclonal antibody orantigen-binding fragment can be conjugated to an agent such as, forexample, a cytotoxic agent, a small molecule, a hormone, an enzyme, agrowth factor, a cytokine, a ribozyme, a peptidomimetic, a chemical, aprodrug, a nucleic acid molecule including coding sequences (such asantisense, RNAi, gene-targeting constructs, etc.), or a detectable label(e.g., an NMR or X-ray contrasting agent, fluorescent molecule, etc.).In certain embodiments, an anti-C5 antibody or antigen-binding fragment(e.g., Fab, Fv, single-chain scFv, Fab′, and F(ab′)₂) is linked to amolecule that increases the half-life of the antibody or antigen-bindingfragment (see above).

Several possible vector systems are available for the expression ofcloned heavy chain and light chain genes in mammalian cells. One classof vectors relies upon the integration of the desired gene sequencesinto the host cell genome. Cells which have stably integrated DNA can beselected by simultaneously introducing drug resistance genes such as E.coli gpt (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA, 78:2072)or Tn5 neo (Southern and Berg (1982) Mol. Appl. Genet. 1:327). Theselectable marker gene can be either linked to the DNA gene sequences tobe expressed, or introduced into the same cell by co-transfection(Wigler et al. (1979) Cell 16:77). A second class of vectors utilizesDNA elements which confer autonomously replicating capabilities to anextrachromosomal plasmid. These vectors can be derived from animalviruses, such as bovine papillomavirus (Sarver et al. (1982) Proc. Natl.Acad. Sci. USA, 79:7147), polyoma virus (Deans et al. (1984) Proc. Natl.Acad. Sci. USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature293:79).

Since an immunoglobulin cDNA is comprised only of sequences representingthe mature mRNA encoding an antibody protein, additional gene expressionelements regulating transcription of the gene and processing of the RNAare required for the synthesis of immunoglobulin mRNA. These elementsmay include splice signals, transcription promoters, including induciblepromoters, enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama and Berg(1983) Mol. Cell. Biol. 3:280; Cepko et al. (1984) Cell 37:1053; andKaufman (1985) Proc. Natl. Acad. Sci. USA 82:689.

As is evident from the disclosure, antibodies that binds to humancomplement components (e.g., antibodies that bind to C5, C5b, or C5a)can be used in therapies (e.g., therapies for a complement-associateddisorder), including combination therapies, as well as in the monitoringof disease progression.

In the therapeutic embodiments of the present disclosure, bispecificantibodies are contemplated. Bispecific antibodies are monoclonal,preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for the human complement componentC5 antigen the other one is for any other antigen.

Methods for making bispecific antibodies are within the purview of thoseskilled in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the co-expression of twoimmunoglobulin heavy-chain/light-chain pairs, where the two heavy chainshave different specificities (Milstein and Cuello (1983) Nature305:537-539). Antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) can be fused toimmunoglobulin constant domain sequences. The fusion preferably is withan immunoglobulin heavy-chain constant domain, including at least partof the hinge, C_(H)2, and C_(H)3 regions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host organism. For further details ofillustrative currently known methods for generating bispecificantibodies see, e.g., Suresh et al. (1986) Methods in Enzymology121:210; PCT Publication No. WO 96/27011; Brennan et al. (1985) Science229:81; Shalaby et al., J. Exp. Med. (1992) 175:217-225; Kostelny et al.(1992) J. Immunol. 148(5):1547-1553; Hollinger et al. (1993) Proc. Natl.Acad. Sci. USA 90:6444-6448; Gruber et al. (1994) J. Immunol. 152:5368;and Tutt et al. (1991) J. Immunol. 147:60. Bispecific antibodies alsoinclude cross-linked or heteroconjugate antibodies. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. (See, e.g., Kostelny et al. (1992) J. Immunol.148(5):1547-1553). The leucine zipper peptides from the Fos and Junproteins may be linked to the Fab′ portions of two different antibodiesby gene fusion. The antibody homodimers may be reduced at the hingeregion to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448 has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a heavy-chain variable domain (VH) connected to alight-chain variable domain (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. (See, e.g., Gruber et al. (1994) J. Immunol. 152:5368.)Alternatively, the antibodies can be “linear antibodies” as describedin, e.g., Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly,these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

The disclosure also embraces variant forms of bispecific antibodies suchas the tetravalent dual variable domain immunoglobulin (DVD-Ig)molecules described in Wu et al. (2007) Nat Biotechnol 25(11):1290-1297.The DVD-Ig molecules are designed such that two different light chainvariable domains (VL) from two different parent antibodies are linked intandem directly or via a short linker by recombinant DNA techniques,followed by the light chain constant domain. Methods for generatingDVD-Ig molecules from two parent antibodies are further described in,e.g., PCT Publication Nos. WO 08/024,188 and WO 07/024,715, thedisclosures of each of which are incorporated herein by reference intheir entirety.

Methods for Treatment

The above-described compositions (e.g., any of the C5 inhibitorsdescribed herein or pharmaceutical compositions thereof) are useful in,inter alfa, methods for treating or preventing a variety ofcomplement-associated disorders (e.g., AP-associated disorders orCP-associated disorders) in a subject. The compositions can beadministered to a subject, e.g., a human subject, using a variety ofmethods that depend, in part, on the route of administration. The routecan be, e.g., intravenous injection or infusion (IV), subcutaneousinjection (SC), intraperitoneal (IP), intrapulmonary, intraocular, orintramuscular injection. Certain inhibitors, e.g., small molecules, canbe orally administered to a subject.

Administration can be achieved by, e.g., local infusion, injection, orby means of an implant. The implant can be of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. The implant can be configured for sustained or periodicrelease of the composition to the subject. (See, e.g., U.S. PatentApplication Publication No. 20080241223; U.S. Pat. Nos. 5,501,856;4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures ofeach of which are incorporated herein by reference in their entirety.)The composition can be delivered to the subject by way of an implantabledevice based on, e.g., diffusive, erodible, or convective systems, e.g.,osmotic pumps, biodegradable implants, electrodiffusion systems,electroosmosis systems, vapor pressure pumps, electrolytic pumps,effervescent pumps, piezoelectric pumps, erosion-based systems, orelectromechanical systems.

A suitable dose of a complement inhibitor (e.g., a C5 inhibitor such asan anti-C5 antibody) described herein, which dose is capable of treatingor preventing a complement-associated disorder in a subject, can dependon a variety of factors including, e.g., the age, sex, and weight of asubject to be treated and the particular inhibitor compound used. Forexample, a different dose of an anti-C5 antibody may be required totreat a subject with RA as compared to the dose of a C5-specific siRNAmolecule that is required to treat the same subject. Other factorsaffecting the dose administered to the subject include, e.g., the typeor severity of the complement-associated disorder. For example, asubject having RA may require administration of a different dosage of ananti-C5 antibody than a subject with AMD. Other factors can include,e.g., other medical disorders concurrently or previously affecting thesubject, the general health of the subject, the genetic disposition ofthe subject, diet, time of administration, rate of excretion, drugcombination, and any other additional therapeutics that are administeredto the subject. It should also be understood that a specific dosage andtreatment regimen for any particular subject will depend upon thejudgment of the treating medical practitioner (e.g., doctor or nurse).

An antibody described herein can be administered as a fixed dose, or ina milligram per kilogram (mg/kg) dose. In some embodiments, the dose canalso be chosen to reduce or avoid production of antibodies or other hostimmune responses against one or more of the active antibodies in thecomposition. While in no way intended to be limiting, exemplary dosagesof an antibody include, e.g., 1-100 μg/kg, 0.5-50 μg/kg, 0.1-100 μg/kg,0.5-25 μg/kg, 1-20 μg/kg, and 1-10 μg/kg, 1-100 mg/kg, 0.5-50 mg/kg,0.1-100 mg/kg, 0.5-25 mg/kg, 1-20 mg/kg, and 1-10 mg/kg. Exemplarydosages of an antibody described herein include, without limitation, 0.1μg/kg, 0.5 μg/kg, 1.0 μg/kg, 2.0 μg/kg, 4 μg/kg, and 8 μg/kg, 0.1 mg/kg,0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4 mg/kg, and 8 mg/kg. Further exemplarydosage amounts and schedules are provided herein (see, e.g., Tables 1and 2).

A pharmaceutical composition can include a therapeutically effectiveamount of a complement inhibitor (e.g., a C5 inhibitor such as ananti-C5 antibody) described herein. Such effective amounts can bereadily determined by one of ordinary skill in the art based, in part,on the effect of the administered antibody, or the combinatorial effectof the antibody and one or more additional active agents, if more thanone agent is used. A therapeutically effective amount of an antibodydescribed herein can also vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody (and one or more additional active agents) to elicit a desiredresponse in the individual, e.g., amelioration of at least one conditionparameter, e.g., amelioration of at least one symptom of thecomplement-associated disorder. For example, a therapeutically effectiveamount of an antibody that binds to C5a and C5b can inhibit (lessen theseverity of or eliminate the occurrence of) and/or prevent a particulardisorder, and/or any one of the symptoms of the particular disorderknown in the art or described herein. A therapeutically effective amountis also one in which any toxic or detrimental effects of the compositionare outweighed by the therapeutically beneficial effects.

Suitable human doses of a C5 inhibitor (e.g., an anti-C5 antibody)described herein can further be evaluated in, e.g., Phase I doseescalation studies. See, e.g., van Gurp et al. (2008) Am JTransplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res13(2, part 1):523-531; and Hetherington et al. (2006) AntimicrobialAgents and Chemotherapy 50(10): 3499-3500.

The terms “therapeutically effective amount” or “therapeuticallyeffective dose,” or similar terms used herein are intended to mean anamount of an agent (e.g., a C5 inhibitor) that will elicit the desiredbiological or medical response (e.g., an improvement in one or moresymptoms of a complement-associated disorder). In some embodiments, acomposition described herein contains a therapeutically effective amountof an anti-C5 antibody. In some embodiments, a composition describedherein contains a therapeutically effective amount of a siRNA, whichspecifically binds to and promotes inactivation of C5 mRNA in amammalian cell. In some embodiments, a composition described hereincontains a therapeutically effective amount of an antibody, whichspecifically binds to C5a. In some embodiments, the composition containsany of the antibodies described herein and one or more (e.g., three,four, five, six, seven, eight, nine, 10, or 11 or more) additionaltherapeutic agents such that the composition as a whole istherapeutically effective. For example, a composition can contain ananti-C5 antibody described herein and an immunosuppressive agent,wherein the antibody and agent are each at a concentration that whencombined are therapeutically effective for treating or preventing acomplement-associated disorder in a subject.

Toxicity and therapeutic efficacy of such compositions can be determinedby known pharmaceutical procedures in cell cultures or experimentalanimals (e.g., animal models of any of the complement-associateddisorders described herein). These procedures can be used, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. A complementinhibitor (e.g., a C5 inhibitor such as an anti-C5 antibody, an anti-C5aantibody, or a nucleic acid that binds to and promotes the inactivationof C5 mRNA in a mammalian cell) that exhibits a high therapeutic indexis preferred. While compositions that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue and to minimize potentialdamage to normal cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch an inhibitor lies generally within a range of circulatingconcentrations of the inhibitor that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For a C5inhibitor (e.g., an anti-C5 antibody or an anti-C5a antibody) used asdescribed herein (e.g., for treating or preventing acomplement-associated disorder), the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography or byELISA.

In some embodiments, the methods can be performed in conjunction withother therapies for complement-associated disorders. For example, thecomposition can be administered to a subject at the same time, prior to,or after, plasmapheresis, IVIG therapy, plasma infusion, or plasmaexchange. See, e.g., Appel et al. (2005) J. Am. Soc Nephrol.16:1392-1404. In some embodiments, a C5 inhibitor (e.g., an anti-C5antibody or an anti-C5a antibody) described herein is not administeredin conjunction with WIG. In some embodiments, the composition can beadministered to a subject at the same time, prior to, or after, a kidneytransplant. Exemplary methods for transplanting an organ (e.g., akidney) or tissue along with exemplary dosing schedules for an anti-C5antibody are provided herein.

A “subject,” as used herein, can be any mammal. For example, a subjectcan be a human (e.g., a patient), a non-human primate (e.g., monkey,baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, acat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. Insome embodiments, the subject is an infant (e.g., a human infant). Insome embodiments, the subject is a female.

As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment(such as treatment with a composition comprising a complement inhibitor(e.g., a C5 inhibitor such as an anti-C5 antibody, an anti-C5a antibody,or a nucleic acid (e.g., an siRNA or antisense nucleic acid) that bindsto and promotes the inactivation of a C5 mRNA in a mammalian cell).

As described above, the complement inhibitors (e.g., a C5 inhibitor suchas an anti-C5 antibody) described herein can be used to treat a varietyof complement-associated disorders such as, e.g., AP-associateddisorders and/or CP-associated disorders. Such disorders include,without limitation, rheumatoid arthritis (RA); antiphospholipid antibodysyndrome; lupus nephritis; ischemia-reperfusion injury; atypicalhemolytic uremic syndrome (aHUS); typical or infectious hemolytic uremicsyndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica(NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS);macular degeneration (e.g., age-related macular degeneration (AMD));hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome;thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss;Pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss;and traumatic brain injury. (See, e.g., Holers (2008) ImmunologicalReviews 223:300-316 and Holers and Thurman (2004) Molecular Immunology41:147-152.) In some embodiments, the complement-associated disorder isa complement-associated vascular disorder such as a cardiovasculardisorder, myocarditis, a cerebrovascular disorder, a peripheral (e.g.,musculoskeletal) vascular disorder, a renovascular disorder, amesenteric/enteric vascular disorder, vasculitis, Henoch-Schönleinpurpura nephritis, systemic lupus erythematosus-associated vasculitis,vasculitis associated with rheumatoid arthritis, immune complexvasculitis, Takayasu's disease, dilated cardiomyopathy, diabeticangiopathy, Kawasaki's disease (arteritis), venous gas embolus (VGE),and restenosis following stent placement, rotational atherectomy, andpercutaneous transluminal coronary angioplasty (PTCA). (See, e.g., U.S.patent application publication no. 20070172483.) Additionalcomplement-associated disorders include, without limitation, MG, CAD,dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease,systemic inflammatory response sepsis, septic shock, spinal cord injury,glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis,pemphigus, AIHA, ITP, Goodpasture syndrome, Degos disease,antiphospholipid syndrome (APS), and catastrophic APS (CAPS).

As used herein, a subject “at risk for developing acomplement-associated disorder” (e.g., an AP-associated disorder or aCP-associated disorder) is a subject having one or more (e.g., two,three, four, five, six, seven, or eight or more) risk factors fordeveloping the disorder. Risk factors will vary depending on theparticular complement-associated disorder, but are well known in the artof medicine. For example, risk factors for developing DDD include, e.g.,a predisposition to develop the condition, i.e., a family history of thecondition or a genetic predisposition to develop the condition such as,e.g., one or more mutations in the gene encoding complement factorH(CFH), complement factor H-related 5 (CFHR5), and/or complementcomponent C3 (C3). Such DDD-associated mutations as well as methods fordetermining whether a subject carries one or more of the mutations areknown in the art and described in, e.g., Licht et al. (2006) Kidney Int.70:42-50; Zipfel et al. (2006) “The role of complement inmembranoproliferative glomerulonephritis,” In: Complement and KidneyDisease, Springer, Berlin, pages 199-221; Ault et al. (1997) J Biol.Chem. 272:25168-75; Abrera-Abeleda et al. (2006) J Med. Genet.43:582-589; Poznansky et al. (1989) J Immunol. 143:1254-1258; Jansen etal. (1998) Kidney Int. 53:331-349; and Hegasy et al. (2002) Am J Pathol161:2027-2034. Thus, a human at risk for developing DDD can be, e.g.,one who has one or more DDD-associated mutations in the gene encodingCFH or one with a family history of developing the disease.

Risk factors for TTP are well known in the art of medicine and include,e.g., a predisposition to develop the condition, i.e., a family historyof the condition or a genetic predisposition to develop the conditionsuch as, e.g., one or more mutations in the ADAMTS13 gene. ADAMTS13mutations associated with TTP are reviewed in detail in, e.g., Levy etal. (2001) Nature 413:488-494; Kokame et al. (2004) Semin. Hematol.41:34-40; Licht et al. (2004) Kidney Int. 66:955-958; and Noris et al.(2005) J. Am. Soc. Nephrol. 16:1177-1183. Risk factors for TTP alsoinclude those conditions or agents that are known to precipitate TTP, orTTP recurrence, such as, but not limited to, cancer, bacterialinfections (e.g., Bartonella sp. infections), viral infections (e.g.,HIV and Kaposi's sarcoma virus), pregnancy, or surgery. See, e.g., Averyet al. (1998) American Journal of Hematology 58:148-149 and Tsai,supra). TTP, or recurrence of TTP, has also been associated with the useof certain therapeutic agents (drugs) including, e.g., ticlopidine,FK506, corticosteroids, tamoxifen, or cyclosporin A (see, e.g., Gordonet al. (1997) Seminars in Hematology 34(2):140-147). Hereinafter, suchmanifestations of TTP may be, where appropriate, referred to as, e.g.,“infection-associated TTP,” “pregnancy-associated TTP,” or“drug-associated TTP.” Thus, a human at risk for developing TTP can be,e.g., one who has one or more TTP-associated mutations in the ADAMTS13gene. A human at risk for developing a recurrent form of TTP can be one,e.g., who has had TTP and has an infection, is pregnant, or isundergoing surgery.

Risk factors for aHUS are well known in the art of medicine and include,e.g., a predisposition to develop the condition, i.e., a family historyof the condition or a genetic predisposition to develop the conditionsuch as, e.g., one or more mutations in complement Factor H(CFH),membrane cofactor protein (MCP; CD46), C4b-binding protein, complementfactor B (CFB), or complement factor I (CFI). (See, e.g., Warwicker etal. (1998) Kidney Int. 53:836-844; Richards et al. (2001) Am J HumGenet. 68:485-490; Caprioli et al. (2001) Am Soc Nephrol 12:297-307;Neuman et al. (2003) J Med Genet. 40:676-681; Richards et al. (2006)Proc Natl Acad Sci USA 100:12966-12971; Fremeaux-Bacchi et al. (2005) JAm Soc Nephrol 17:2017-2025; Esparza-Gordillo et al. (2005) Hum MolGenet. 14:703-712; Goicoechea de Jorge et al. (2007) Proc Natl Acad SciUSA 104(1):240-245; Blom et al. (2008) J Immunol. 180(9):6385-91; andFremeaux-Bacchi et al. (2004) J Medical Genet. 41:e84). (See alsoKavanagh et al. (2006) supra.) Risk factors also include, e.g.,infection with Streptococcus pneumoniae, pregnancy, cancer, exposure toanti-cancer agents (e.g., quinine, mitomycin C, cisplatin, orbleomycin), exposure to immunotherapeutic agents (e.g., cyclosporine,OKT3, or interferon), exposure to anti-platelet agents (e.g.,ticlopidine or clopidogrel), HIV infection, transplantation, autoimmunedisease, and combined methylmalonic aciduria and homocystinuria (cblC).See, e.g., Constantinescu et al. (2004) Am J Kidney Dis 43:976-982;George (2003) Curr Opin Hematol 10:339-344; Gottschall et al. (1994) AmJ Hematol 47:283-289; Valavaara et al. (1985) Cancer 55:47-50; Miralbellet al. (1996) J Clin Oncol 14:579-585; Dragon-Durey et al. (2005) J AmSoc Nephrol 16:555-63; and Becker et al. (2004) Clin Infect Dis39:S267-S275.

Risk factors for HELLP are well known in the art of medicine andinclude, e.g., multiparous pregnancy, maternal age over 25 years,Caucasian race, the occurrence of preeclampsia or HELLP in a previouspregnancy, and a history of poor pregnancy outcome. (See, e.g., Sahin etal. (2001) Nagoya Med J 44(3):145-152; Sullivan et al. (1994) Am JObstet Gynecol 171:940-943; and Padden et al. (1999) Am Fam Physician60(3):829-836.) For example, a pregnant, Caucasian woman who developedpreeclampsia during a first pregnancy can be one at risk for developingHELLP syndrome during, or following, a second pregnancy.

Risk factors for CAD are well known in the art of medicine and include,e.g., conditions or agents that are known to precipitate CAD, or CADrecurrence, such as, but not limited to, neoplasms or infections (e.g.,bacterial and viral infections). Conditions known to be associated withthe development of CAD include, e.g., HIV infection (and AIDS),hepatitis C infection, Mycoplasma pneumonia infection, Epstein-Ban virus(EBV) infection, cytomegalovirus (CMV) infection, rubella, or infectiousmononucleosis. Neoplasms associated with CAD include, withoutlimitation, non-Hodgkin's lymphoma. Hereinafter, such manifestations ofCAD may be, where appropriate, referred to as, e.g.,“infection-associated CAD” or “neoplasm-associated CAD.” Thus, a humanat risk for developing CAD can be, e.g., one who has an HIV infection,rubella, or a lymphoma. See also, e.g., Gertz (2006) Hematology 1:19-23;Horwitz et al. (1977) Blood 50:195-202; Finland and Barnes (1958) AMAArch Intern Med 191:462-466; Wang et al. (2004) Acta Paediatr Taiwan45:293-295; Michaux et al. (1998) Ann Hematol 76:201-204; and Chang etal. (2004) Cancer Genet Cytogenet 152:66-69.

Risk factors for a thrombotic microangiopathy (TMA) are well known inthe art of medicine and include, e.g., a medical history of aHUS, TTP,or other conditions that are associated with TMA such as lupus, cancers,disseminating intravascular coagulation and other coagulopathies, andpre-eclampsia. See, e.g., Copelovitch and Kaplan (2008) Pediatr Nephrol23(10):1761-7.

Risk factors for PCH are well known in the art of medicine and include,e.g., conditions or agents that are known to precipitate PCH, or PCHrecurrence, such as, but not limited to, neoplasms, infections (e.g.,bacterial and viral infections), or certain immunizations (e.g., measlesimmunization). Conditions known to be associated with the development ofPCH include, e.g., syphilis (a Treponema palladium infection), measles,mumps, influenza virus infection, varicella-zoster virus infection,cytomegalovirus (CMV) infection, Epstein-Barr virus (EBV) infection,adenovirus infection, parvovirus B19 infection, Coxsackie A9 infection,Haemophilus influenza infection, Mycoplasma pneumoniae infection, andKlebsiella pneumoniae infection. See, e.g., Bunch et al. (1972) Arch DisChild 47(252):299-300; Ziman et al. (2004) Transfusion 44(8):1127-1128;Sokol et al. (1984) Acta Haematol 72(4): 245-257; Papalia et al. (2000)Br J Haematol 109(2): 328-9; Sokol et al. (1982) Acta Haematol68(4):268-277; and Bell et al. (1973) Transfusion 13(3):138-141.Neoplasms associated with PCH include, without limitation, both solidand hematopoietic neoplasms such as myelofibrosis, chronic lymphocyticleukemia (CLL), and non-Hodgkin's lymphoma. See, e.g., Sharara et al.(1994) South Med J. 87(3):397-9; Sivakumaran et al. (1999) Br J Haematol105(1): 278-9; Breccia et al. (2004) Eur J Haematol 73(4):304-6; andWynn et al. (1998) Clin Lab Haematol 20(6):373-5. Hereinafter, suchmanifestations of PCH may be, where appropriate, referred to as, e.g.,“infection-associated PCH” or “neoplasm-associated PCH.” Thus, a humanat risk for developing PCH can be, e.g., one who has an EBV infection ora lymphoma.

Risk factors for MG are well known in the art of medicine and include,e.g., a predisposition to develop the condition, i.e., a family historyof the condition or a genetic predisposition to develop the conditionsuch as familial MG. For example, some HLA types are associated with anincreased risk for developing MG. Risk factors for MG include theingestion or exposure to certain MG-inducing drugs such as, but notlimited to, D-penicillamine. See, e.g., Drosos et al. (1993) Clin ExpRheumatol. 11(4):387-91 and Kaeser et al. (1984) Acta Neurol ScandSuppl. 100:39-47. As MG can be episodic, a subject who has previouslyexperienced one or more symptoms of having MG can be at risk forrelapse. Thus, a human at risk for developing MG can be, e.g., one whohas a family history of MG and/or one who has ingested or beenadministered an MG-inducing drug such as D-penicillamine.

As used herein, a subject “at risk for developing CAPS” is a subjecthaving one or more (e.g., two, three, four, five, six, seven, or eightor more) risk factors for developing the disorder. Approximately 60% ofthe incidences of CAPS are preceded by a precipitating event such as aninfection. Thus, risk factors for CAPS include those conditions known toprecipitate CAPS such as, but not limited to, certain cancers (e.g.,gastric cancer, ovarian cancer, lymphoma, leukemia, endometrial cancer,adenocarcinoma, and lung cancer), pregnancy, puerperium,transplantation, primary APS, rheumatoid arthritis (RA), systemic lupuserythematosus (SLE), surgery (e.g., eye surgery), and certaininfections. Infections include, e.g., parvovirus B19 infection andhepatitis C infection. Hereinafter, such manifestations of CAPS may bereferred to as, e.g., “cancer-associated CAPS,”“transplantation-associated CAPS,” “RA-associated CAPS,”“infection-associated CAPS,” or “SLE-associated CAPS.” See, e.g.,Soltész et al. (2000) Haematologia (Budep) 30(4):303-311; Ideguchi etal. (2007) Lupus 16(1):59-64; Manner et al. (2008) Am J Med. Sci.335(5):394-7; Miesbach et al. (2006) Autoimmune Rev. 6(2):94-7;Gómez-Puerta et al. (2006) Autoimmune Rev. 6(2):85-8; Gömez-Puerta etal. (2006) Semin. Arthritis Rheum. 35(5):322-32; Kasamon et al. (2005)Haematologia 90(3):50-53; Atherson et al. (1998) Medicine 77(3):195-207;and Canpolat et al. (2008) Clin Pediatr 47(6):593-7. Thus, a human atrisk for developing CAPS can be, e.g., one who has primary CAPS and/or acancer that is known to be associated with CAPS.

From the above it will be clear that subjects “at risk for developing acomplement-associated disorder” (e.g., an AP-associated disorder or aCP-associated disorder) are not all the subjects within a species ofinterest.

A subject “suspected of having a complement-associated disorder” (e.g.,an alternative complement pathway-associated disorder) is one having oneor more (e.g., two, three, four, five, six, seven, eight, nine, or 10 ormore) symptoms of the disease. Symptoms of these disorders will varydepending on the particular disorder, but are known to those of skill inthe art of medicine. For example, symptoms of DDD include, e.g.: one orboth of hematuria and proteinuria; acute nephritic syndrome; drusendevelopment and/or visual impairment; acquired partial lipodystrophy andcomplications thereof; and the presence of serum C3 nephritic factor(C3NeF), an autoantibody directed against C3bBb, the C3 convertase ofthe alternative complement pathway. (See, e.g., Appel et al. (2005),supra). Symptoms of aHUS include, e.g., severe hypertension,proteinuria, uremia, lethargy/fatigue, irritability, thrombocytopenia,microangiopathic hemolytic anemia, and renal function impairment (e.g.,acute renal failure). Symptoms of TTP include, e.g., microthrombi,thrombocytopenia, fever, low ADAMTS13 metalloproteinase expression oractivity, fluctuating central nervous system abnormalities, renalfailure, microangiopathic hemolytic anemia, bruising, purpura, nauseaand vomiting (e.g., resulting from ischemia in the GI tract or fromcentral nervous system involvement), chest pain due to cardiac ischemia,seizures, and muscle and joint pain. Symptoms of RA can include, e.g.,stiffness, swelling, fatigue, anemia, weight loss, fever, and often,crippling pain. Some common symptoms of rheumatoid arthritis includejoint stiffness upon awakening that lasts an hour or longer; swelling ina specific finger or wrist joints; swelling in the soft tissue aroundthe joints; and swelling on both sides of the joint. Swelling can occurwith or without pain, and can worsen progressively or remain the samefor years before progressing. Symptoms of HELLP are known in the art ofmedicine and include, e.g., malaise, epigastric pain, nausea, vomiting,headache, right upper quadrant pain, hypertension, proteinuria, blurredvision, gastrointestinal bleeding, hypoglycemia, paresthesia, elevatedliver enzymes/liver damage, anemia (hemolytic anemia), and low plateletcount, any of which in combination with pregnancy or recent pregnancy.(See, e.g., Tomsen (1995) Am J Obstet Gynecol 172:1876-1890; Sibai(1986) Am J Obstet Gynecol 162:311-316; and Padden (1999), supra.)

Symptoms of CAPS are well known in the art of medicine and include,e.g., histopathological evidence of multiple small vessel occlusions;the presence of antiphospholipid antibodies (usually at high titer),vascular thromboses, severe multi-organ dysfunction, malignanthypertension, acute respiratory distress syndrome, disseminatedintravascular coagulation, microangiopathic hemolytic anemia,schistocytes, and thrombocytopenia. CAPS can be distinguished from APSin that patients with CAPS generally present with severe multiple organdysfunction or failure, which is characterized by rapid, diffuse smallvessel ischemia and thromboses predominantly affecting the parenchymalorgans. In contrast, APS is associated with single venous or arterialmedium-to-large blood vessel occlusions. Symptoms of MG include, e.g.,fatigability and a range of muscle weakness-related conditionsincluding: ptosis (of one or both eyes), diplopia, unstable gait,depressed or distorted facial expressions, and difficulty chewing,talking, or swallowing. In some instances, a subject can present withpartial or complete paralysis of the respiratory muscles. Symptoms ofCAD include, e.g., pain, fever, pallor, anemia, reduced blood flow tothe extremities (e.g., with gangrene), and renal disease or acute renalfailure. In some embodiments, the symptoms can occur following exposureto cold temperatures.

From the above it will be clear that subjects “suspected of having acomplement-associated disorder” are not all the subjects within aspecies of interest.

In some embodiments, the methods can include identifying the subject asone having, suspected of having, or at risk for developing, acomplement-associated disorder in a subject. Suitable methods foridentifying the subject are known in the art. For example, suitablemethods (e.g., sequencing techniques or use of microarrays) fordetermining whether a human subject has a DDD-associated mutation in aCFH, CFHR5, or C3 gene are described in, e.g., Licht et al. (2006)Kidney Int. 70:42-50; Zipfel et al. (2006), supra; Ault et al. (1997) J.Biol. Chem. 272:25168-75; Abrera-Abeleda et al. (2006) J Med Genet.43:582-589; Poznansky et al. (1989) J Immunol. 143:1254-1258; Jansen etal. (1998) Kidney Int. 53:331-349; and Hegasy et al. (2002) Am J Pathol161:2027-2034. Methods for detecting the presence of characteristicDDD-associated electron-dense deposits are also well known in the art.For example, a medical practitioner can obtain a tissue biopsy from thekidney of a patient and subject the tissue to electron microscopy. Themedical practitioner may also examine the tissue by immunofluorescenceto detect the presence of C3 using an anti-C3 antibody and/or lightmicroscopy to determine if there is membranoproliferativeglomerulonephritis. See, e.g., Walker et al. (2007) Mod. Pathol.20:605-616 and Habib et al. (1975) Kidney Int. 7:204-215. In someembodiments, the identification of a subject as one having DDD caninclude assaying a blood sample for the presence of C3NeF. Methods fordetecting the presence of C3NeF in blood are described in, e.g.,Schwertz et al. (2001) Pediatr Allergy Immunol. 12:166-172.

In some embodiments, the medical practitioner can determine whetherthere is increased complement activation in a subject's serum. Indiciaof increased complement activation include, e.g., a reduction in CH50, adecrease in C3, and an increase in C3dg/C3d. See, e.g., Appel et al.(2005), supra. In some embodiments, a medical practitioner can examine asubject's eye for evidence of the development of drusen and/or othervisual pathologies such as AMD. For example, a medical practitioner canuse tests of retinal function such as, but not limited to, darkadaptation, electroretinography, and electrooculography (see, e.g.,Colville et al. (2003) Am J Kidney Dis. 42:E2-5).

Methods for identifying a subject as one having, suspected of having, orat risk for developing, TTP are also known in the art. For example,Miyata et al. describe a variety of assays for measuring ADAMTS13activity in a biological sample obtained from a subject (Curr OpinHematol (2007) 14(3):277-283). Suitable ADAMTS13 activity assays, aswell as phenotypically normal ranges of ADAMTS13 activity in a humansubject, are described in, e.g., Tsai (2003) J. Am. Soc. Nephrol14:1072-1081; Furlan et al. (1998) New Engl J Med. 339:1578-1584;Matsumoto et al. (2004) Blood 103:1305-1310; and Mori et al. (2002)Transfusion 42:572-580. Methods for detecting the presence of inhibitorsof ADAMTS13 (e.g., autoantibodies that bind to ADAMTS13) in a biologicalsample obtained from a subject are known in the art. For example, aserum sample from a patient can be mixed with a serum sample from asubject without TTP to detect the presence of anti-ADAMTS13 antibodies.In another example, immunoglobulin protein can be isolated from patientserum and used in in vitro ADAMTS13 activity assays to determine if ananti-ADAMTS13 antibody is present. See, e.g., Dong et al. (2008) Am JHematol. 83(10):815-817. In some embodiments, risk of developing TTP canbe determined by assessing whether a patient carries one or moremutations in the ADAMTS13 gene. Suitable methods (e.g., nucleic acidarrays or DNA sequencing) for detecting a mutation in the ADAMTS13 geneare known in the art and described in, e.g., Levy et al., supra; Kokameet al., supra; Licht et al., supra; and Noris et al., supra.

In addition, methods for identifying a subject as one having, suspectedof having, or at risk for developing aHUS are known in the art. Forexample, laboratory tests can be performed to determine whether a humansubject has thrombocytopenia, microangiopathic hemolytic anemia, oracute renal insufficiency. Thrombocytopenia can be diagnosed by amedical professional as one or more of: (i) a platelet count that isless than 150,000/mm³ (e.g., less than 60,000/mm³); (ii) a reduction inplatelet survival time, reflecting enhanced platelet disruption in thecirculation; and (iii) giant platelets observed in a peripheral smear,which is consistent with secondary activation of thrombocytopoiesis.Microangiopathic hemolytic anemia can be diagnosed by a medicalprofessional as one or more of: (i) hemoglobin concentrations that areless than 10 mg/dL (e.g., less than 6.5 mg/dL); (ii) increased serumlactate dehydrogenase (LDH) concentrations (>460 U/L); (iii)hyperbilirubinemia, reticulocytosis, circulating free hemoglobin, andlow or undetectable haptoglobin concentrations; and (iv) the detectionof fragmented red blood cells (schistocytes) with the typical aspect ofburr or helmet cells in the peripheral smear together with a negativeCoombs test. (See, e.g., Kaplan et al. (1992) “Hemolytic Uremic Syndromeand Thrombotic Thrombocytopenic Purpura,” Informa Health Care (ISBN0824786637) and Zipfel (2005) “Complement and Kidney Disease,” Springer(ISBN 3764371668).)

A subject can also be identified as having aHUS by evaluating bloodconcentrations of C3 and C4 as a measure of complement activation ordysregulation. In addition, as is clear from the foregoing disclosure, asubject can be identified as having genetic aHUS by identifying thesubject as harboring one or more mutations in a gene associated withaHUS such as CFI, CFB, CFH, or MCP (supra). Suitable methods fordetecting a mutation in a gene include, e.g., DNA sequencing and nucleicacid array techniques. (See, e.g., Breslin et al. (2006) Clin Am SocNephrol 1:88-99 and Goicoechea de Jorge et al. (2007) Proc Natl Acad SciUSA 104:240-245.)

Symptoms characteristic of TMA include, e.g., fever, microangiopathichemolytic anemia (schistocytes in a blood smear), renal failure,thrombocytopenia, and neurological manifestations.

Methods for diagnosing a subject as one having, suspected of having, orat risk for developing, RA are also known in the art of medicine. Forexample, a medical practitioner can examine the small joints of thehands, wrists, feet, and knees to identify inflammation in a symmetricaldistribution. The practitioner may also perform a number of tests toexclude other types of joint inflammation including arthritis due toinfection or gout. In addition, rheumatoid arthritis is associated withabnormal antibodies in the blood circulation of afflicted patients. Forexample, an antibody referred to as “rheumatoid factor” is found inapproximately 80% of patients. In another example, anti-citrullineantibody is present in many patients with rheumatoid arthritis and thusit is useful in the diagnosis of rheumatoid arthritis when evaluatingpatients with unexplained joint inflammation. See, e.g., van Venrooij etal. (2008) Ann NY Acad Sci 1143:268-285 and Habib et al. (2007) ImmunolInvest 37(8):849-857. Another antibody called “the antinuclear antibody”(ANA) is also frequently found in patients with rheumatoid arthritis.See, e.g., Benucci et al. (2008) Clin Rheumatol 27(1):91-95; Julkunen etal. (2005) Scan J Rheumatol 34(2):122-124; and Miyawaki et al. (2005) JRheumatol 32(8):1488-1494.

A medical practitioner can also examine red blood cell sedimentationrate to help in diagnosing RA in a subject. The sedimentation rate canbe used as a crude measure of the inflammation of the joints and isusually faster during disease flares and slower during remissions.Another blood test that can be used to measure the degree ofinflammation present in the body is the C-reactive protein.

Furthermore, joint x-rays can also be used to diagnose a subject ashaving rheumatoid arthritis. As RA progresses, the x-rays can show bonyerosions typical of rheumatoid arthritis in the joints. Joint x-rays canalso be helpful in monitoring the progression of disease and jointdamage over time. Bone scanning, a radioactive test procedure, candemonstrate the inflamed joints.

Methods for identifying a subject as one having, suspected of having, orat risk for developing, HELLP are known in the art of medicine. Hallmarksymptoms of HELLP syndrome include hemolysis, elevated liver enzymes,and low platelet count. Thus, a variety of tests can be performed onblood from a subject to determine the level of hemolysis, theconcentration of any of a variety of liver enzymes, and the plateletlevel in the blood. For example, the presence of schistocytes and/orelevated free hemoglobin, bilirubin, or serum LDH levels is anindication of intravascular hemolysis. Routine laboratory testing can beused to determine the platelet count as well as the blood level of liverenzymes such as, but not limited to, aspartate aminotransferase (AST)and alanine transaminase (ALT). Suitable methods for identifying asubject as having HELLP syndrome are also described in, e.g., Sibai etal. (1993), supra; Martin et al. (1990), supra; Padden (1999), supra;and Gleicher and Buttino (1998) “Principles & Practice of MedicalTherapy in Pregnancy,” 3^(rd) Edition, Appleton & Lange (ISBN083857677X).

Suitable methods for identifying the subject as having MG can bequalitative or quantitative. For example, a medical practitioner canexamine the status of a subject's motor functions using a physicalexamination. Other qualitative tests include, e.g., an ice-pack test,wherein an ice pack is applied to a subject's eye (in a case of ocularMG) to determine if one or more symptoms (e.g., ptosis) are improved bycold (see, e.g., Sethi et al. (1987) Neurology 37(8):1383-1385). Othertests include, e.g., the “sleep test,” which is based on the tendencyfor MG symptoms to improve following rest. In some embodiments,quantitative or semi-quantitative tests can be employed by a medicalpractitioner to determine if a subject has, is suspected of having, oris at risk for developing, MG. For example, a medical practitioner canperform a test to detect the presence or amount of MG-associatedautoantibodies in a serum sample obtained from a subject. MG-associatedautoantibodies include, e.g., antibodies that bind to, and modulate theactivity of, acetylcholine receptor (AChR), muscle-specific receptortyrosine kinase (MuSK), and/or striational protein. (See, e.g.,Conti-Fine et al. (2006), supra). Suitable assays useful for detectingthe presence or amount of an MG-associated antibody in a biologicalsample are known in the art and described in, e.g., Hoch et al. (2001)Nat Med 7:365-368; Vincent et al. (2004) Semin Neurol. 24:125-133;McConville et al. (2004) Ann. Neurol. 55:580-584; Boneva et al. (2006) JNeuroimmunol. 177:119-131; and Romi et al. (2005) Arch Neurol.62:442-446.

Additional methods for diagnosing MG include, e.g., electrodiagnostictests (e.g., single-fiber electromyography) and the Tensilon (oredrophonium) test, which involves injecting a subject with theacetylcholinesterase inhibitor edrophonium and monitoring the subjectfor an improvement in one or more symptoms. See, e.g., Pascuzzi (2003)Semin Neurol 23(1):83-88; Katirji et al. (2002) Neurol Clin 20:557-586;and “Guidelines in Electrodiagnostic Medicine. American Association ofElectrodiagnostic Medicine,” Muscle Nerve 15:229-253.

A subject can be identified as having CAD using an assay to detect thepresence or amount (titer) of agglutinating autoantibodies that bind tothe I antigen on red blood cells. The antibodies can be monoclonal(e.g., monoclonal IgM or IgA) or polyclonal. Suitable methods fordetecting these antibodies are described in, e.g., Christenson and Dacie(1957) Br J Haematol 3:153-164 and Christenson et al. (1957) Br JHaematol 3:262-275. A subject can also be diagnosed as having CAD usingone or more of a complete blood cell count (CBC), urinalysis,biochemical studies, and a Coombs test to test for hemolysis in blood.For example, biochemical studies can be used to detect elevated lactasedehydrogenase levels, elevated unconjugated bilirubin levels, lowhaptoglobin levels, and/or the presence of free plasma hemoglobin, allof which can be indicative of acute hemolysis. Other tests that can beused to detect CAD include detecting complement levels in the serum. Forexample, due to consumption during the acute phase of hemolysis,measured plasma complement levels (e.g., C2, C3, and C4) are decreasedin CAD.

Typical (or infectious) HUS, unlike aHUS, is often identifiable by aprodrome of diarrhea, often bloody in nature, which results frominfection with a shiga-toxin producing microorganism. A subject can beidentified as having typical HUS when shiga toxins and/or serumantibodies against shiga toxin or LPS are detected in the stool of anindividual. Suitable methods for testing for anti-shiga toxin antibodiesor LPS are known in the art. For example, methods for detectingantibodies that bind to shiga toxins Stx1 and Stx2 or LPS in humans aredescribed in, e.g., Ludwig et al. (2001) J Clin Microbiol39(6):2272-2279.

Symptoms of this condition are known to those of skill in the art ofmedicine and include, e.g., pain, fever, pallor, icterus, urticarialdermal eruption, hemoglobinuria, hemoglobinemia, anemia, and renaldisease or acute renal failure. In some embodiments, the symptoms canoccur following exposure to cold temperatures.

In some embodiments, the methods can include identifying the subject asone having, suspected of having, or at risk for developing, PCH.Suitable methods for identifying the subject are known in the art. Forexample, a subject can be diagnosed as having PCH using aDonath-Landsteiner test, which is an assay to detect the presence of theDonath-Landsteiner antibody in a subject's serum. The procedure involvesincubating three samples—(1) the subject's serum; (2) normal serum; and(3) a mix of the subject's serum and normal serum—with P-antigenexpressing red blood cells at 0 to 4° C. Next, the sample is warmed to37° C. and visually inspected for hemolysis. If the Donath-Landsteinerantibody is present, hemolysis should occur in samples (1) and (3), butnot in (2). See, e.g., Funato et al. (2007) Eur J Haematol 79(5):462;Win et al. (2005) Transfus Med. 15(3):254; Sokol et al. (1998)Immunohematology 14(3):109-12; Eder (2005) Immunohematology 21(2):56-62;and Dacie et al. (1957) Br J Haematol 3:77-87. A subject can also bediagnosed as having PCH using one or more of a complete blood cell count(CBC), urinalysis, biochemical studies, and a Coombs test. For example,biochemical studies can be used to detect elevated lactase dehydrogenaselevels, elevated unconjugated bilirubin levels, low haptoglobin levels,and/or the presence of free plasma hemoglobin, all of which can beindicative of acute hemolysis. Other tests that can be used to detectPCH include detecting complement levels in the serum. For example, dueto consumption during the acute phase of hemolysis, measured plasmacomplement levels (e.g., C2, C3, and C4) are decreased in PCH. See also,e.g., Nordhagen et al. (1984) Acta Paediatr Scand 73(2):258-262;Lindgren et al. (1985) Transfusion 25(2):142-4; Nordhagen et al. (1991)Transfusion 31(2):190-1; and Garratty (2001) Transfusion 41(8):1073-4.

In some embodiments, the composition can be administered to a subjectprophylactically to prevent, or prevent relapse or recurrence of, PCH.For example, a subject who previously had an advanced Mycoplasmainfection or who is newly diagnosed with a PCH-associated neoplasm canbe administered a composition described herein to prevent, lessen theseverity of, or prevent a recurrence of PCH.

In some embodiments, a C5 inhibitor (e.g., an anti-C5 antibody)described herein can be administered to a subject as a monotherapy.Alternatively, as described above, the antibody can be administered to asubject as a combination therapy with another treatment, e.g., anothertreatment for DDD, TTP, aHUS, RA, HELLP, MG, CAD, CAPS, tHUS, Degosdisease, or any other complement-associated disorder described herein.For example, the combination therapy can include administering to thesubject (e.g., a human patient) one or more additional agents (e.g.,anti-coagulants, anti-hypertensives, or corticosteroids) that provide atherapeutic benefit to the subject who has, or is at risk of developing,DDD. In some embodiments, the combination therapy can includeadministering to the subject (e.g., a human patient) a C5 inhibitor(e.g., an anti-C5 antibody or an anti-C5a antibody) and animmunosuppressive agent such as Remicade® for use in treating RA. Insome embodiments, the C5 inhibitor and the one or more additional activeagents are administered at the same time. In other embodiments, a C5inhibitor is administered first in time and the one or more additionalactive agents are administered second in time. In some embodiments, theone or more additional active agents are administered first in time andthe C5 inhibitor is administered second in time.

A C5 inhibitor (e.g., an anti-C5 antibody) described herein can replaceor augment a previously or currently administered therapy. For example,upon treating with an anti-C5 antibody, administration of the one ormore additional active agents can cease or diminish, e.g., beadministered at lower levels. In some embodiments, administration of theprevious therapy can be maintained. In some embodiments, a previoustherapy will be maintained until the level of the C5 inhibitor (e.g.,anti-C5 antibody or an anti-C5a antibody) reaches a level sufficient toprovide a therapeutic effect. The two therapies can be administered incombination.

In some embodiments, a C5 inhibitor can be administered to a patientchronically. For example, a patient chronically treated with acomplement-inhibiting agent (e.g., a C5 inhibitor or a C5a inhibitor)can be treated for a period of greater than or equal to 2 weeks (e.g.,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years or for theremainder of the patient's life) with the agent in an amount and with adosing frequency that are sufficient to maintain a concentration of theagent in the patient's blood that inhibits or substantially inhibitssystemic complement activity in the patient. To maintain systemiccomplement inhibition in a patient, a C5 inhibitor can be chronicallyadministered to the patient, e.g., once a week, once every two weeks,twice a week, once a day, once a month, or once every three weeks. Insome embodiments of any of the methods described herein, the C5inhibitor can be administered to a patient in an amount and with afrequency of administration effective to maintain a concentration of atleast: 0.7 (e.g., at least 0.8, 0.9, one, two, three, four, five, six,seven, eight, nine, or 10 or more) bivalent C5 inhibitor (e.g., a wholeantibody) molecule(s) per every C5 molecule in the patient's blood; or1.5 (e.g., at least 1.6, 1.7, 1.8, 1.9, two, three, four, five, six,seven, eight, nine, or 10 or more) monovalent C5 inhibitor (e.g., asingle chain anti-C5 antibody or a Fab fragment of the antibody)molecule(s) per every C5 molecule in the patient's blood. For example,in some embodiments a monovalent anti-C5 antibody (e.g., a single chainantibody or a Fab antibody fragment) can be administered to a patient inan amount and with a frequency effective to maintain a concentration ofat least 1.5 (e.g., at least 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, or 7 ormore) monovalent anti-C5 antibodies per C5 molecule in the blood. Insome embodiments of any of the methods described herein, an anti-C5antibody is administered to the patient in an amount and with afrequency that are effective to maintain a concentration of at least 40(e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90,95, 100, 110, or 120 or more) μg of the antibody per milliliter of thepatient's blood. Exemplary chronic dosing strategies are describedherein (see, e.g., Tables 1 and 2).

In some embodiments, the C5 inhibitor (or C5a inhibitor) can beadministered to a subject even after one or more symptoms have beenameliorated. Monitoring a subject (e.g., a human patient) for animprovement in a complement-associated disorder, as defined herein,means evaluating the subject for a change in a disease parameter, e.g.,an improvement in one or more symptoms of the disease. Such symptomsinclude any of the symptoms of complement-associated disorders describedherein. In some embodiments, the evaluation is performed at least 1hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day,2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after anadministration. The subject can be evaluated in one or more of thefollowing periods: prior to beginning of treatment; during thetreatment; or after one or more elements of the treatment have beenadministered. Evaluating can include evaluating the need for furthertreatment, e.g., evaluating whether a dosage, frequency ofadministration, or duration of treatment should be altered. It can alsoinclude evaluating the need to add or drop a selected therapeuticmodality, e.g., adding or dropping any of the treatments for any of thecomplement-associated disorders described herein.

In some embodiments, the complement inhibitor can be chronicallyadministered to a patient in need thereof in an amount and with afrequency that are effective to reduce and maintain serum hemolyticactivity at less than or equal to 20 (e.g., 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, or even below 5) %. See, e.g., Hill et al.(2005) Blood 106(7):2559. In some embodiments, the complement inhibitorcan be administered to a patient in an amount and with a frequency thatare effective to maintain serum lactate dehydrogenase (LDH) levels atwithin at least 20 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6, or even below 5) % of the normal range for LDH. See Hill et al.(2005) supra. In some embodiments, the complement inhibitor isadministered to the patient in an amount and with a frequency that areeffective to maintain a serum LDH level less than 550 (e.g., less than540, 530, 520, 510, 500, 490, 480, 470, 450, 440, 430, 420, 410, 400, orless than 300) IU/L. In some embodiments, administration (e.g., chronicadministration) of a C5 inhibitor (e.g., an anti-C5 antibody such aseculizumab) or a C5a inhibitor (e.g., an anti-C5a antibody) results inamelioration of one or more of a patient's symptoms to within 40 (e.g.,39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22,21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,or even 1) % of its normal level or value. For example, in someembodiments, the elevated blood pressure in an aHUS patient treated(e.g., chronically treated) with an anti-C5 antibody can be reduced to alevel that is within 40% of the level that is normal for a person of thepatient's age, race, height, weight, sex, and physical health.

In some embodiments, the complement inhibitor (e.g., a C5 inhibitor orC5a inhibitor) is administered to a subject even after the patient hasentered clinical remission. Determining clinical remission of acomplement-associated disorder is well within the skill set of theskilled artisan in medicine. For example, elements determinative ofclinical remission for aHUS are described in, e.g., Nürnberger et al.(2009) N Engl J Med 360(5):542-544. Clinical remission for CAPS isdescribed in, e.g., Manner et al. (2008) Am J Med Sci 335(5):394-397.

The disclosure also provides methods for allogeneic organ or tissuetransplantation. The method includes transplanting an organ or tissueinto a patient in need thereof, wherein prior to and following thetransplanting a C5 inhibitor is administered to the patient in an amountand with a frequency effective to substantially inhibit systemiccomplement activity in the patient. As described herein, the C5inhibitor (e.g., the anti-C5 antibody) can be administered in an amountand with a frequency to maintain a concentration of at least one C5inhibitor molecule (e.g., at least one anti-C5 antibody) per C5 moleculein the patient's blood. In some embodiments, a monovalent anti-C5antibody (e.g., a single chain antibody or a Fab antibody fragment) canbe administered to a patient in an amount with a frequency effective tomaintain a concentration of at least 1.5 (e.g., at least 1.6, 1.7, 1.8,1.9, 2, 3, 4, 5, 6, or 7 or more) monovalent anti-C5 antibodies per C5molecule in the blood. In some embodiments, the C5 inhibitor (e.g., theanti-C5 antibody) can be administered to the patient in an amount andwith a frequency to maintain a concentration of at least at least 40(e.g., 41, 42, 43, 44, 45, 46, 47, 48, 48, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90,95, 100, 110, or 120 or more) μg of the inhibitor (e.g., the anti-C5antibody) in the patient's blood. In some embodiments, at least 800(e.g., at least 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, or 1200 or more) mgof the anti-C5 antibody (e.g., eculizumab) is administered to thepatient less than 24 (e.g., less than 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less than 2) hours priorto transplanting the organ or tissue to the patient. In someembodiments, the methods can also include, prior to the transplanting,contacting (e.g., soaking) the donor organ or tissue with a C5 inhibitor(e.g., an anti-C5 antibody such as eculizumab) for an amount of time andunder conditions that inhibit complement activation in the organ ortissue upon transplantation. The organ can be, e.g., skin, a kidney,heart, lung, limb (e.g., finger or toe), or liver. In some embodiments,the methods can include administering a C5 inhibitor (e.g., an anti-C5antibody) to the donor patient prior to removal of the organ or tissuefor transplant. The patient can have, be at risk for developing, or besuspected of having aHUS. In some embodiments, at least 700 (e.g., atleast 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, 1100, or 1200 or more) mg of the anti-C5 antibody isadministered to the patient less than 24 (e.g., less than 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or lessthan 2) hours following the transplanting. In some embodiments, theanti-C5 antibody is chronically administered to the patient followingthe transplanting. For example, the anti-C5 antibody can be chronicallyadministered to the patient for at least 9 weeks (e.g., 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, or 52 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 10.5, or 12 years or for the remainder of the patient'slife) under the following dosing schedule: at least 700 (e.g., at least710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,990, 1000, 1100, or 1200 or more) mg of the anti-C5 antibody less than24 hours after transplanting the organ or tissue; at least 700 (e.g., atleast 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, 1000, 1100, or 1200 or more) mg of the anti-C5 antibody onceper week for four weeks after the initial post-transplant dose; at least700 (e.g., at least 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,950, 960, 970, 980, 990, 1000, 1100, or 1200 or more) mg of the anti-C5antibody once during the fifth week; and at least 700 (e.g., at least710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,990, 1000, 1100, or 1200 or more) mg of the anti-C5 antibody bi-weeklythereafter for the remainder of the dosing schedule. In a preferredembodiment, the anti-C5 antibody is administered such that the firstfour doses are at least 900 mg of the antibody; 1200 mg is administeredon the fifth week; and 1200 mg is administered to the patient bi-weeklythereafter for the remainder of the chronic treatment schedule. Additionexemplary dosing schedules are provided in Tables 1 and 2.

In some embodiments, the methods include administering animmunosuppressant to the patient. Suitable immunosuppressants for use inthe methods include, but are not limited to, ATG or ALG, OKT3,daclizumab, basiliximab, corticosteroids, 15-deoxyspergualin,cyclosporins, tacrolimus, azathioprine, methotrexate, mycophenolatemofetil, 6-mercaptopurine, bredinin, brequinar, leflunamide,cyclophosphamide, sirolimus, anti-CD4 monoclonal antibodies, CTLA4-Ig,anti-CD154 monoclonal antibodies, anti-LFA1 monoclonal antibodies,anti-LFA-3 monoclonal antibodies, anti-CD2 monoclonal antibodies, andanti-CD45 antibodies.

Types of organs or tissues that can be transplanted using the methodsdescribed herein include, e.g., heart, kidney, lung, pancreas, liver,vascular tissue, eye, cornea, lens, skin, bone marrow, muscle,connective tissue, gastrointestinal tissue, nervous tissue, bone, stemcells, islets, cartilage, hepatocytes, and hematopoietic cells.

In some embodiments, the transplant methods will result in prolongationof the graft in the recipient patient by at least one month (e.g.,three, four, five, six, seven, eight, nine, 10, 11, or 12 months or 1,2, 3, 4, 5, 6, 7, 8, 9, or even 10 years or more).

Ex Vivo Approaches.

An ex vivo strategy for treating or preventing a complement-associateddisorder (e.g., an AP-associated disorder or a CP-associated disorder)can involve transfecting or transducing one or more cells obtained froma subject with a polynucleotide encoding a complement inhibitor (e.g.,an anti-C5 antibody, anti-C5a antibody, or a nucleic acid (e.g., asiRNA) that binds to and promotes inactivation of a C5 mRNA in amammalian cell) described herein. For example, the cells can betransfected with a single vector encoding a heavy and light chain of anantibody that binds to C5 protein, or the cells can be transfected witha first vector encoding a heavy chain and a second vector encoding alight chain of the antibody.

The transfected or transduced cells are then returned to the subject.The cells can be any of a wide range of types including, withoutlimitation, hemopoietic cells (e.g., bone marrow cells, macrophages,monocytes, dendritic cells, T cells, or B cells), fibroblasts,epithelial cells, endothelial cells, keratinocytes, or muscle cells.Such cells can act as a source (e.g., sustained or periodic source) ofthe C5 inhibitor (e.g., anti-C5 antibody, anti-C5a antibody, or nucleicacid (above)) for as long as they survive in the subject. In someembodiments, the vectors and/or cells can be configured for inducible orrepressible expression of the C5 inhibitor (see, e.g., Schockett et al.(1996) Proc Natl Acad Sci USA 93: 5173-5176 and U.S. Pat. No.7,056,897).

Preferably, the cells are obtained from the subject (autologous), butcan potentially be obtained from a subject of the same species otherthan the subject (allogeneic).

Suitable methods for obtaining cells from a subject and transducing ortransfecting the cells are known in the art of molecular biology. Forexample, the transduction step can be accomplished by any standard meansused for ex vivo gene therapy, including calcium phosphate, lipofection,electroporation, viral infection (see above), and biolistic genetransfer. (See, e.g., Sambrook et al. (supra) and Ausubel et al. (1992)“Current Protocols in Molecular Biology,” Greene Publishing Associates.)Alternatively, liposomes or polymeric microparticles can be used. Cellsthat have been successfully transduced can be selected, for example, forexpression of the coding sequence or of a drug resistance gene.

Kits

The disclosure also features articles of manufacture or kits, whichinclude a container with a label; and a composition containing one ormore complement inhibitors described herein. For example, the kit cancontain one or more of an anti-C5a antibody, an anti-C5 antibody, and anucleic acid (e.g., an siRNA or antisense nucleic acid) that binds toand promotes inactivation of a C5 mRNA in a mammalian cell. The labelindicates that the composition is to be administered to a subject (e.g.,a human) having, suspected of having, or at risk for developing, acomplement-associated disorder (e.g., an AP- or CP-associated disorder)such as DDD, aHUS, TTP, HELLP, RA, AMD, tHUS, MG, CAD, PCH, CAPS, Degosdisease, or any other complement pathway-associated disorder describedherein. The kit can, optionally, include a means for administering thecomposition to the subject. For example, the kits can include one ormore syringes.

In some embodiments, the kits can further include one or more additionalactive agents such as any of those described herein. For example, thekits can include one or more corticosteroids, anti-hypertensives,immunosuppressives, and anti-seizure agents.

The following examples are intended to illustrate, not limit, theinvention.

Example 1

A human adult patient is identified by a medical practitioner as havingan inherited form of aHUS. Once a week for four weeks the patient isadministered a composition containing eculizumab at a dose of 900 mg.The patient then receives at least 1200 mg of eculizumab once during thefifth week and at least 1200 mg of eculizumab bi-weekly thereafter. Thepatient and medical practitioner observe a substantial improvement in atleast two known symptoms of aHUS during the initial treatment.Eculizumab is chronically administered to the patient even after themedical practitioner determines that the aHUS is in remission.

Example 2

A human patient weighing around 25 kg is identified by a medicalpractitioner as having aHUS. Once a week for two weeks the patient isadministered a composition containing eculizumab at a dose of at least600 mg. The patient then receives at least 600 mg of eculizumab onceduring the third week and at least 600 mg of eculizumab bi-weeklythereafter. The patient and medical practitioner observe a substantialimprovement in at least two known symptoms of aHUS during the initialtreatment. Eculizumab is chronically administered to the patient evenafter the medical practitioner determines that the aHUS is in remissionin order to prevent a recurrence of aHUS in the patient.

Example 3

A human patient weighing around 35 kg is identified by a medicalpractitioner as having CAPS. Once a week for two weeks the patient isadministered a composition containing eculizumab at a dose of at least600 mg. The patient then receives at least 900 mg of eculizumab onceduring the third week and at least 900 mg of eculizumab bi-weeklythereafter. The patient and medical practitioner observe a substantialimprovement in at least two known symptoms of CAPS during the initialtreatment. Eculizumab is chronically administered to the patient evenafter the medical practitioner determines that the CAPS is in remissionin order to prevent, or substantially reduce the likelihood of, arecurrence of CAPS in the patient.

Example 4

A human patient weighing around 7 kg is identified by a medicalpractitioner as having aHUS. The patient has TMA in her kidneys as aresult of the aHUS. For one week the patient is administered acomposition containing eculizumab at a dose of at least 300 mg. Thepatient then receives at least 300 mg of eculizumab once during thesecond week and at least 300 mg of eculizumab every three weeksthereafter. The patient and medical practitioner observe a substantialimprovement in at least two known symptoms of aHUS during the initialtreatment. The medical practitioner also observes that the TMA in thepatient's kidneys resolves and no new TMA occurs while the patient isbeing chronically administered eculizumab. Eculizumab is chronicallyadministered to the patient even after the medical practitionerdetermines that the aHUS is in remission in order to prevent, orsubstantially reduce the likelihood of, a recurrence of aHUS in thepatient and any further damage to her kidneys that could result fromrecurrence.

Example 5

A human patient in need of a kidney transplant is intravenouslyadministered eculizumab at a dose of 1200 mg less than 24 hours beforethe transplant operation. An allogeneic kidney is transplanted into thepatient. Less than 24 hours after the kidney transplant, the patient isadministered another 1200 mg of eculizumab. Once a week for four weeksfollowing the first post-operation dose of eculizumab, the patientreceives 900 mg of eculizumab. The patient receives 1200 mg ofeculizumab on the fifth week after the initial post-operation dose ofeculizumab and then is maintained on a dosing schedule that includes1200 mg of eculizumab bi-weekly thereafter. The medical practitionerobserves a substantial improvement in the survival of the transplantedkidney in the patient.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

What is claimed is:
 1. A method for treating atypical hemolytic uremicsyndrome (aHUS), the method comprising administering to a patient inneed thereof eculizumab in an amount effective to treat aHUS in thepatient; wherein the eculizumab is intravenously administered to thepatient under the following schedule: at least 600 mg of eculizumab onceper week for four consecutive weeks; and beginning at week five,maintenance doses of at least 900 mg eculizumab every two weeksthereafter.
 2. The method of claim 1, wherein the eculizumab isadministered to the patient in an amount and with a frequency tomaintain at least 50 μg of eculizumab per milliliter of the patient'sblood.
 3. The method of claim 1, wherein the eculizumab is administeredto the patient in an amount and with a frequency to maintain at least100 μg of eculizumab per milliliter of the patient's blood.
 4. Themethod of claim 1, wherein the eculizumab is administered to the patientin an amount and with a frequency to maintain a concentration of atleast 0.7 molecules of eculizumab per every C5 molecule in the patient'sblood.